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Sample records for novo sequence analysis

  1. Top-down analysis of protein samples by de novo sequencing techniques

    Energy Technology Data Exchange (ETDEWEB)

    Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.; VanDuijn, Martijn M.; Liu, Xiaowen; Tolić, Nikola; Luider, Theo M.; Paša-Tolić, Ljiljana; Pevzner, Pavel A.

    2016-05-14

    MOTIVATION: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. RESULTS: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns.

  2. Characterization of Liaoning cashmere goat transcriptome: sequencing, de novo assembly, functional annotation and comparative analysis.

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    Hongliang Liu

    Full Text Available BACKGROUND: Liaoning cashmere goat is a famous goat breed for cashmere wool. In order to increase the transcriptome data and accelerate genetic improvement for this breed, we performed de novo transcriptome sequencing to generate the first expressed sequence tag dataset for the Liaoning cashmere goat, using next-generation sequencing technology. RESULTS: Transcriptome sequencing of Liaoning cashmere goat on a Roche 454 platform yielded 804,601 high-quality reads. Clustering and assembly of these reads produced a non-redundant set of 117,854 unigenes, comprising 13,194 isotigs and 104,660 singletons. Based on similarity searches with known proteins, 17,356 unigenes were assigned to 6,700 GO categories, and the terms were summarized into three main GO categories and 59 sub-categories. 3,548 and 46,778 unigenes had significant similarity to existing sequences in the KEGG and COG databases, respectively. Comparative analysis revealed that 42,254 unigenes were aligned to 17,532 different sequences in NCBI non-redundant nucleotide databases. 97,236 (82.51% unigenes were mapped to the 30 goat chromosomes. 35,551 (30.17% unigenes were matched to 11,438 reported goat protein-coding genes. The remaining non-matched unigenes were further compared with cattle and human reference genes, 67 putative new goat genes were discovered. Additionally, 2,781 potential simple sequence repeats were initially identified from all unigenes. CONCLUSION: The transcriptome of Liaoning cashmere goat was deep sequenced, de novo assembled, and annotated, providing abundant data to better understand the Liaoning cashmere goat transcriptome. The potential simple sequence repeats provide a material basis for future genetic linkage and quantitative trait loci analyses.

  3. De novo sequencing and assembly analysis of transcriptome in the Sodom apple (Calotropis gigantea)

    National Research Council Canada - National Science Library

    Muriira, Nkatha G; Xu, Wei; Muchugi, Alice; Xu, Jianchu; Liu, Aizhong

    2015-01-01

    .... In this study, using the Illumina high throughput sequencing technique we obtained about 45 million paired end sequencing reads, De novo assembled and generated a total of 133,634 transcripts with a mean of 1837.47 bp in length...

  4. Transcriptome sequencing and de novo analysis of the copepod Calanus sinicus using 454 GS FLX.

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    Juan Ning

    Full Text Available BACKGROUND: Despite their species abundance and primary economic importance, genomic information about copepods is still limited. In particular, genomic resources are lacking for the copepod Calanus sinicus, which is a dominant species in the coastal waters of East Asia. In this study, we performed de novo transcriptome sequencing to produce a large number of expressed sequence tags for the copepod C. sinicus. RESULTS: Copepodid larvae and adults were used as the basic material for transcriptome sequencing. Using 454 pyrosequencing, a total of 1,470,799 reads were obtained, which were assembled into 56,809 high quality expressed sequence tags. Based on their sequence similarity to known proteins, about 14,000 different genes were identified, including members of all major conserved signaling pathways. Transcripts that were putatively involved with growth, lipid metabolism, molting, and diapause were also identified among these genes. Differentially expressed genes related to several processes were found in C. sinicus copepodid larvae and adults. We detected 284,154 single nucleotide polymorphisms (SNPs that provide a resource for gene function studies. CONCLUSION: Our data provide the most comprehensive transcriptome resource available for C. sinicus. This resource allowed us to identify genes associated with primary physiological processes and SNPs in coding regions, which facilitated the quantitative analysis of differential gene expression. These data should provide foundation for future genetic and genomic studies of this and related species.

  5. Transcriptome sequencing and De Novo analysis of Youngia japonica using the illumina platform.

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    Yulan Peng

    Full Text Available Youngia japonica, a weed species distributed worldwide, has been widely used in traditional Chinese medicine. It is an ideal plant for studying the evolution of Asteraceae plants because of its short life history and abundant source. However, little is known about its evolution and genetic diversity. In this study, de novo transcriptome sequencing was conducted for the first time for the comprehensive analysis of the genetic diversity of Y. japonica. The Y. japonica transcriptome was sequenced using Illumina paired-end sequencing technology. We produced 21,847,909 high-quality reads for Y. japonica and assembled them into contigs. A total of 51,850 unigenes were identified, among which 46,087 were annotated in the NCBI non-redundant protein database and 41,752 were annotated in the Swiss-Prot database. We mapped 9,125 unigenes onto 163 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database. In addition, 3,648 simple sequence repeats (SSRs were detected. Our data provide the most comprehensive transcriptome resource currently available for Y. japonica. C4 photosynthesis unigenes were found in the biological process of Y. japonica. There were 5596 unigenes related to defense response and 1344 ungienes related to signal transduction mechanisms (10.95%. These data provide insights into the genetic diversity of Y. japonica. Numerous SSRs contributed to the development of novel markers. These data may serve as a new valuable resource for genomic studies on Youngia and, more generally, Cichoraceae.

  6. Sequencing, de novo assembly and comparative analysis of Raphanus sativus transcriptome.

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    Wu, Gang; Zhang, Libin; Yin, Yongtai; Wu, Jiangsheng; Yu, Longjiang; Zhou, Yanhong; Li, Maoteng

    2015-01-01

    Raphanus sativus is an important Brassicaceae plant and also an edible vegetable with great economic value. However, currently there is not enough transcriptome information of R. sativus tissues, which impedes further functional genomics research on R. sativus. In this study, RNA-seq technology was employed to characterize the transcriptome of leaf tissues. Approximately 70 million clean pair-end reads were obtained and used for de novo assembly by Trinity program, which generated 68,086 unigenes with an average length of 576 bp. All the unigenes were annotated against GO and KEGG databases. In the meanwhile, we merged leaf sequencing data with existing root sequencing data and obtained better de novo assembly of R. sativus using Oases program. Accordingly, potential simple sequence repeats (SSRs), transcription factors (TFs) and enzyme codes were identified in R. sativus. Additionally, we detected a total of 3563 significantly differentially expressed genes (DEGs, P = 0.05) and tissue-specific biological processes between leaf and root tissues. Furthermore, a TFs-based regulation network was constructed using Cytoscape software. Taken together, these results not only provide a comprehensive genomic resource of R. sativus but also shed light on functional genomic and proteomic research on R. sativus in the future.

  7. De novo peptide sequencing by deep learning.

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    Tran, Ngoc Hieu; Zhang, Xianglilan; Xin, Lei; Shan, Baozhen; Li, Ming

    2017-07-18

    De novo peptide sequencing from tandem MS data is the key technology in proteomics for the characterization of proteins, especially for new sequences, such as mAbs. In this study, we propose a deep neural network model, DeepNovo, for de novo peptide sequencing. DeepNovo architecture combines recent advances in convolutional neural networks and recurrent neural networks to learn features of tandem mass spectra, fragment ions, and sequence patterns of peptides. The networks are further integrated with local dynamic programming to solve the complex optimization task of de novo sequencing. We evaluated the method on a wide variety of species and found that DeepNovo considerably outperformed state of the art methods, achieving 7.7-22.9% higher accuracy at the amino acid level and 38.1-64.0% higher accuracy at the peptide level. We further used DeepNovo to automatically reconstruct the complete sequences of antibody light and heavy chains of mouse, achieving 97.5-100% coverage and 97.2-99.5% accuracy, without assisting databases. Moreover, DeepNovo is retrainable to adapt to any sources of data and provides a complete end-to-end training and prediction solution to the de novo sequencing problem. Not only does our study extend the deep learning revolution to a new field, but it also shows an innovative approach in solving optimization problems by using deep learning and dynamic programming.

  8. The first complete chloroplast genome sequences of Ulmus species by de novo sequencing: Genome comparative and taxonomic position analysis

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    Zhang, Shuang; Yu, Xiao-Yue; Ren, Ya-Chao; Yang, Min-Sheng; Wang, Jin-Mao

    2017-01-01

    Elm (Ulmus) has a long history of use as a high-quality heavy hardwood famous for its resistance to drought, cold, and salt. It grows in temperate, warm temperate, and subtropical regions. This is the first report of Ulmaceae chloroplast genomes by de novo sequencing. The Ulmus chloroplast genomes exhibited a typical quadripartite structure with two single-copy regions (long single copy [LSC] and short single copy [SSC] sections) separated by a pair of inverted repeats (IRs). The lengths of the chloroplast genomes from five Ulmus ranged from 158,953 to 159,453 bp, with the largest observed in Ulmus davidiana and the smallest in Ulmus laciniata. The genomes contained 137–145 protein-coding genes, of which Ulmus davidiana var. japonica and U. davidiana had the most and U. pumila had the fewest. The five Ulmus species exhibited different evolutionary routes, as some genes had been lost. In total, 18 genes contained introns, 13 of which (trnL-TAA+, trnL-TAA−, rpoC1-, rpl2-, ndhA-, ycf1, rps12-, rps12+, trnA-TGC+, trnA-TGC-, trnV-TAC-, trnI-GAT+, and trnI-GAT) were shared among all five species. The intron of ycf1 was the longest (5,675bp) while that of trnF-AAA was the smallest (53bp). All Ulmus species except U. davidiana exhibited the same degree of amplification in the IR region. To determine the phylogenetic positions of the Ulmus species, we performed phylogenetic analyses using common protein-coding genes in chloroplast sequences of 42 other species published in NCBI. The cluster results showed the closest plants to Ulmaceae were Moraceae and Cannabaceae, followed by Rosaceae. Ulmaceae and Moraceae both belonged to Urticales, and the chloroplast genome clustering results were consistent with their traditional taxonomy. The results strongly supported the position of Ulmaceae as a member of the order Urticales. In addition, we found a potential error in the traditional taxonomies of U. davidiana and U. davidiana var. japonica, which should be confirmed with a

  9. Sequencing and de novo analysis of Crassostrea angulata (Fujian oyster from 8 different developing phases using 454 GSFlx.

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    Ji Qin

    Full Text Available Research on the mechanism for early development of shellfish, such as body plan, shell formation, settlement and metamorphosis is currently an active research field. However, studies were still limited and not deep enough because of the lack of genomic resources such as genome or transcriptome sequences. In the present research, de novo transcriptome sequencing was performed for Crassostrea angulata, the most economically important cultured oyster species in China, at eight early developmental stages using the 454 sequencing technology. A total of 555,215 reads were produced with an average length of 309 nucleotides that were then assembled into 10,462 contigs. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Six unique sequences related to settlement, metamorphosis and growth were subsequently analyzed by real-time PCR. Given the lack of whole genome information for oysters, transcriptome and de novo analysis of C. angulata from the eight different developing phases will provide important and useful information on early development mechanism and help genetic breeding of shellfish.

  10. RNA-Seq analysis of Cocos nucifera: transcriptome sequencing and de novo assembly for subsequent functional genomics approaches.

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    Haikuo Fan

    Full Text Available BACKGROUND: Cocos nucifera (coconut, a member of the Arecaceae family, is an economically important woody palm grown in tropical regions. Despite its agronomic importance, previous germplasm assessment studies have relied solely on morphological and agronomical traits. Molecular biology techniques have been scarcely used in assessment of genetic resources and for improvement of important agronomic and quality traits in Cocos nucifera, mostly due to the absence of available sequence information. METHODOLOGY/PRINCIPAL FINDINGS: To provide basic information for molecular breeding and further molecular biological analysis in Cocos nucifera, we applied RNA-seq technology and de novo assembly to gain a global overview of the Cocos nucifera transcriptome from mixed tissue samples. Using Illumina sequencing, we obtained 54.9 million short reads and conducted de novo assembly to obtain 57,304 unigenes with an average length of 752 base pairs. Sequence comparison between assembled unigenes and released cDNA sequences of Cocos nucifera and Elaeis guineensis indicated that the assembled sequences were of high quality. Approximately 99.9% of unigenes were novel compared to the released coconut EST sequences. Using BLASTX, 68.2% of unigenes were successfully annotated based on the Genbank non-redundant (Nr protein database. The annotated unigenes were then further classified using the Gene Ontology (GO, Clusters of Orthologous Groups (COG and Kyoto Encyclopedia of Genes and Genomes (KEGG databases. CONCLUSIONS/SIGNIFICANCE: Our study provides a large quantity of novel genetic information for Cocos nucifera. This information will act as a valuable resource for further molecular genetic studies and breeding in coconut, as well as for isolation and characterization of functional genes involved in different biochemical pathways in this important tropical crop species.

  11. Pseudo-De Novo Assembly and Analysis of Unmapped Genome Sequence Reads in Wild Zebrafish Reveal Novel Gene Content.

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    Faber-Hammond, Joshua J; Brown, Kim H

    2016-04-01

    Zebrafish represents the third vertebrate with an officially completed genome, yet it remains incomplete with additions and corrections continuing with the current release, GRCz10, having 13% of zebrafish cDNA sequences unmapped. This disparity may result from population differences, given that the genome reference was generated from clonal individuals with limited genetic diversity. This is supported by the recent analysis of a single wild zebrafish, which identified over 5.2 million SNPs and 1.6 million in/dels in the previous genome build, zv9. Re-examination of this sequence data set indicated that 13.8% of quality sequence reads failed to align to GRCz10. Using a novel bioinformatics de novo assembly pipeline on these unmappable reads, we identified 1,514,491 novel contigs covering ∼224 Mb of genomic sequence. Among these, 1083 contigs were found to contain a potential gene coding sequence. RNA-seq data comparison confirmed that 362 contigs contained a transcribed DNA sequence, suggesting that a large amount of functional genomic sequence remains unannotated in the zebrafish reference genome. By utilizing the bioinformatics pipeline developed in this study, the zebrafish genome will be bolstered as a model for human disease research. Adaptation of the pipeline described here also offers a cost-efficient and effective method to identify and map novel genetic content across any genome and will ultimately aid in the completion of additional genomes for a broad range of species.

  12. De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

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    Meena, Seema; Kumar, Sarma R.; Venkata Rao, D. K.; Dwivedi, Varun; Shilpashree, H. B.; Rastogi, Shubhra; Shasany, Ajit K.; Nagegowda, Dinesh A.

    2016-01-01

    Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition. PMID:27516768

  13. A combined de novo protein sequencing and cDNA library approach to the venomic analysis of Chinese spider Araneus ventricosus.

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    Duan, Zhigui; Cao, Rui; Jiang, Liping; Liang, Songping

    2013-01-14

    In past years, spider venoms have attracted increasing attention due to their extraordinary chemical and pharmacological diversity. The recently popularized proteomic method highly improved our ability to analyze the proteins in the venom. However, the lack of information about isolated venom proteins sequences dramatically limits the ability to confidently identify venom proteins. In the present paper, the venom from Araneus ventricosus was analyzed using two complementary approaches: 2-DE/Shotgun-LC-MS/MS coupled to MASCOT search and 2-DE/Shotgun-LC-MS/MS coupled to manual de novo sequencing followed by local venom protein database (LVPD) search. The LVPD was constructed with toxin-like protein sequences obtained from the analysis of cDNA library from A. ventricosus venom glands. Our results indicate that a total of 130 toxin-like protein sequences were unambiguously identified by manual de novo sequencing coupled to LVPD search, accounting for 86.67% of all toxin-like proteins in LVPD. Thus manual de novo sequencing coupled to LVPD search was proved an extremely effective approach for the analysis of venom proteins. In addition, the approach displays impeccable advantage in validating mutant positions of isoforms from the same toxin-like family. Intriguingly, methyl esterifcation of glutamic acid was discovered for the first time in animal venom proteins by manual de novo sequencing.

  14. Proteomics-grade de novo sequencing approach

    DEFF Research Database (Denmark)

    Savitski, Mikhail M; Nielsen, Michael L; Kjeldsen, Frank

    2005-01-01

    known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach...... is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65...

  15. De novo Sequencing and Analysis of Lemongrass Transcriptome Provides First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

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    Seema Meena

    2016-07-01

    Full Text Available Aromatic grasses of the genus Cymbopogon (Poaceae family represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavour, fragrance, cosmetic and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step towards understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases (TPS, pyrophosphatases (PPase, alcohol dehydrogenases (ADH, aldo-keto reductases (AKR, carotenoid cleavage dioxygenases (CCD, alcohol acetyltransferases (AAT and aldehyde dehydrogenases (ALDH, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes and acetates. Molecular modeling and docking further supported the role of identified enzymes in aroma formation in Cymbopogon. Also, simple sequence repeats (SSRs were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

  16. De Novo Sequencing and Assembly Analysis of the Pseudostellaria heterophylla Transcriptome

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    Li, Jun; Zhen, Wei; Long, Dengkai; Ding, Ling; Gong, Anhui; Xiao, Chenghong; Jiang, Weike; Liu, Xiaoqing; Zhou, Tao; Huang, Luqi

    2016-01-01

    Pseudostellaria heterophylla (Miq.) Pax is a mild tonic herb widely cultivated in the Southern part of China. The tuberous roots of P. heterophylla accumulate high levels of secondary metabolism products of medicinal value such as saponins, flavonoids, and isoquinoline alkaloids. Despite numerous studies on the pharmacological importance and purification of these compounds in P. heterophylla, their biosynthesis is not well understood. In the present study, we used Illumina HiSeq 4000 sequencing platform to sequence the RNA from flowers, leaves, stem, root cortex and xylem tissues of P. heterophylla. We obtained 616,413,316 clean reads that we assembled into 127, 334 unique sequences with an N50 length of 951 bp. Among these unigenes, 53,184 unigenes (41.76%) were annotated in a public database and 39, 795 unigenes were assigned to 356 KEGG pathways; 23,714 unigenes (8.82%) had high homology with the genes from Beta vulgaris. We discovered 32, 095 DEGs in different tissues and performed GO and KEGG enrichment analysis. The most enriched KEGG pathway of secondary metabolism showed up-regulated expression in tuberous roots as compared with the ground parts of P. heterophylla. Moreover, we identified 72 candidate genes involved in triterpenoids saponins biosynthesis in P. heterophylla. The expression profiles of 11 candidate unigenes were analyzed by quantitative real-time PCR (RT-qPCR). Our study established a global transcriptome database of P. heterophylla for gene identification and regulation. We also identified the candidate unigenes involved in triterpenoids saponins biosynthesis. Our results provide an invaluable resource for the secondary metabolites and physiological processes in different tissues of P. heterophylla. PMID:27764127

  17. De novo sequencing, assembly and analysis of eight different transcriptomes from the Malayan pangolin.

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    Mohamed Yusoff, Aini; Tan, Tze King; Hari, Ranjeev; Koepfli, Klaus-Peter; Wee, Wei Yee; Antunes, Agostinho; Sitam, Frankie Thomas; Rovie-Ryan, Jeffrine Japning; Karuppannan, Kayal Vizi; Wong, Guat Jah; Lipovich, Leonard; Warren, Wesley C; O'Brien, Stephen J; Choo, Siew Woh

    2016-09-13

    Pangolins are scale-covered mammals, containing eight endangered species. Maintaining pangolins in captivity is a significant challenge, in part because little is known about their genetics. Here we provide the first large-scale sequencing of the critically endangered Manis javanica transcriptomes from eight different organs using Illumina HiSeq technology, yielding ~75 Giga bases and 89,754 unigenes. We found some unigenes involved in the insect hormone biosynthesis pathway and also 747 lipids metabolism-related unigenes that may be insightful to understand the lipid metabolism system in pangolins. Comparative analysis between M. javanica and other mammals revealed many pangolin-specific genes significantly over-represented in stress-related processes, cell proliferation and external stimulus, probably reflecting the traits and adaptations of the analyzed pregnant female M. javanica. Our study provides an invaluable resource for future functional works that may be highly relevant for the conservation of pangolins.

  18. Sequencing, de novo assembly, functional annotation and analysis of Phyllanthus amarus leaf transcriptome using the Illumina platform

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    Aparupa eBose Mazumdar

    2016-01-01

    Full Text Available Phyllanthus amarus Schum. & Thonn., a widely distributed annual medicinal herb has a long history of use in the traditional system of medicine for over 2000 years. However, the lack of genomic data for P. amarus, a non-model organism hinders research at the molecular level. In the present study, high-throughput sequencing technology has been employed to enhance better understanding of this herb and provide comprehensive genomic information for future work. Here P. amarus leaf transcriptome was sequenced using the Illumina Miseq platform. We assembled 85,927 non-redundant unitranscript sequences with an average length of 1548 bp, from 18,060,997 raw reads. Sequence similarity analyses and annotation of these unitranscripts were performed against databases like green plants non-redundant (nr protein database, Gene Ontology (GO, Clusters of Orthologous Groups (COG, PlnTFDB, KEGG databases. As a result, 69,394 GO terms, 583 enzyme codes, 134 KEGG maps and 59 Transcription Factor families were generated. Functional and comparative analyses of assembled unitranscripts were also performed with the most closely related species like Populus trichocarpa and Ricinus communis using TRAPID. KEGG analysis showed that a number of assembled unitranscripts were involved in secondary metabolites, mainly phenylpropanoid, flavonoid, terpenoids, alkaloids and lignan biosynthetic pathways that have significant medicinal attributes. Further, Fragments Per Kilobase of transcript per Million mapped reads (FPKM values of the identified secondary metabolite pathway genes were determined and Reverse Transcription PCR (RT-PCR of few of these genes were performed to validate the de novo assembled leaf transcriptome dataset. In addition 65,273 simple sequence repeats (SSRs were also identified. To the best of our knowledge this is the first transcriptomic dataset of P. amarus till date. Our study provides the largest genetic resource that will lead to drug development and

  19. De Novo Transcriptome Sequencing and Analysis of the Cereal Cyst Nematode, Heterodera avenae

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    Kumar, Mukesh; Gantasala, Nagavara Prasad; Roychowdhury, Tanmoy; Thakur, Prasoon Kumar; Banakar, Prakash; Shukla, Rohit N.; Jones, Michael G. K.; Rao, Uma

    2014-01-01

    The cereal cyst nematode (CCN, Heterodera avenae) is a major pest of wheat (Triticum spp) that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptome analysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy) revealed fewer glycoside hydrolases (GHs) but more glycosyl transferases (GTs) and carbohydrate esterases (CEs) when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million) value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb) for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction. PMID:24802510

  20. De novo transcriptome sequencing and analysis of the cereal cyst nematode, Heterodera avenae.

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    Mukesh Kumar

    Full Text Available The cereal cyst nematode (CCN, Heterodera avenae is a major pest of wheat (Triticum spp that reduces crop yields in many countries. Cyst nematodes are obligate sedentary endoparasites that reproduce by amphimixis. Here, we report the first transcriptome analysis of two stages of H. avenae. After sequencing extracted RNA from pre parasitic infective juvenile and adult stages of the life cycle, 131 million Illumina high quality paired end reads were obtained which generated 27,765 contigs with N50 of 1,028 base pairs, of which 10,452 were annotated. Comparative analyses were undertaken to evaluate H. avenae sequences with those of other plant, animal and free living nematodes to identify differences in expressed genes. There were 4,431 transcripts common to H. avenae and the free living nematode Caenorhabditis elegans, and 9,462 in common with more closely related potato cyst nematode, Globodera pallida. Annotation of H. avenae carbohydrate active enzymes (CAZy revealed fewer glycoside hydrolases (GHs but more glycosyl transferases (GTs and carbohydrate esterases (CEs when compared to M. incognita. 1,280 transcripts were found to have secretory signature, presence of signal peptide and absence of transmembrane. In a comparison of genes expressed in the pre-parasitic juvenile and feeding female stages, expression levels of 30 genes with high RPKM (reads per base per kilo million value, were analysed by qRT-PCR which confirmed the observed differences in their levels of expression levels. In addition, we have also developed a user-friendly resource, Heterodera transcriptome database (HATdb for public access of the data generated in this study. The new data provided on the transcriptome of H. avenae adds to the genetic resources available to study plant parasitic nematodes and provides an opportunity to seek new effectors that are specifically involved in the H. avenae-cereal host interaction.

  1. De novo sequencing and comparative analysis of testicular transcriptome from different reproductive phases in freshwater spotted snakehead Channa punctatus

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    Roy, Alivia; Basak, Reetuparna

    2017-01-01

    The spotted snakehead Channa punctatus is a seasonally breeding teleost widely distributed in the Indian subcontinent and economically important due to high nutritional value. The declining population of C. punctatus prompted us to focus on genetic regulation of its reproduction. The present study carried out de novo testicular transcriptome sequencing during the four reproductive phases and correlated differential expression of transcripts with various testicular events in C. punctatus. The Illumina paired-end sequencing of testicular transcriptome from resting, preparatory, spawning and postspawning phases generated 41.94, 47.51, 61.81 and 44.45 million reads, and 105526, 105169, 122964 and 106544 transcripts, respectively. Transcripts annotated using Rattus norvegicus reference protein sequences and classified under various subcategories of biological process, molecular function and cellular component showed that the majority of the subcategories had highest number of transcripts during spawning phase. In addition, analysis of transcripts exhibiting differential expression during the four phases revealed an appreciable increase in upregulated transcripts of biological processes such as cell proliferation and differentiation, cytoskeleton organization, response to vitamin A, transcription and translation, regulation of angiogenesis and response to hypoxia during spermatogenically active phases. The study also identified significant differential expression of transcripts relevant to spermatogenesis (mgat3, nqo1, hes2, rgs4, cxcl2, alcam, agmat), steroidogenesis (star, tkt, gipc3), cell proliferation (eef1a2, btg3, pif1, myo16, grik3, trim39, plbd1), cytoskeletal organization (espn, wipf3, cd276), sperm development (klhl10, mast1, hspa1a, slc6a1, ros1, foxj1, hipk1), and sperm transport and motility (hint1, muc13). Analysis of functional annotation and differential expression of testicular transcripts depending on reproductive phases of C. punctatus helped in

  2. De novo sequencing, assembly and analysis of salivary gland transcriptome of Haemaphysalis flava and identification of sialoprotein genes.

    Science.gov (United States)

    Xu, Xing-Li; Cheng, Tian-Yin; Yang, Hu; Yan, Fen; Yang, Ya

    2015-06-01

    Saliva plays an important role in feeding and pathogen transmission, identification and analysis of tick salivary gland (SG) proteins is considered as a hot spot in anti-tick researching area. Herein, we present the first description of SG transcriptome of Haemaphysalis flava using next-generation sequencing (NGS). A total of over 143 million high-quality reads were assembled into 54,357 unigenes, of which 20,145 (37.06%) had significant similarities to proteins in the Swiss-Prot database. 13,513 annotated sequences were associated with GO terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 14,280 unigenes were assigned to 279 KEGG pathways in total. Reads per kb per million reads (RPKM) analysis showed that there were 3035 down-regulated unigenes and 2260 up-regulated unigenes in the engorged ticks (ET) compared with the semi-engorged one (SET). Several important genes are associated with blood feeding and ingestion as secreted salivary proteins, concluding cysteine, longipain, 4D8, calreticulin, metalloproteases, serine protease inhibitor, enolase, heat shock protein and AV422 in SG, were identified. The qRT-PCR results confirmed that patterns of these genes (except for the longipain gene) expression were consistent with RNA-seq results. This de novo assembly of SG transcriptome of H. flava not only provides more chance for screening and cloning functional genes, but also forms a solid basis for further insight into the changes of salivary proteins during blood-feeding. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Transcriptomic Analysis of Flower Blooming in Jasminum sambac through De Novo RNA Sequencing

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    Yong-Hua Li

    2015-06-01

    Full Text Available Flower blooming is a critical and complicated plant developmental process in flowering plants. However, insufficient information is available about the complex network that regulates flower blooming in Jasminum sambac. In this study, we used the RNA-Seq platform to analyze the molecular regulation of flower blooming in J. sambac by comparing the transcript profiles at two flower developmental stages: budding and blooming. A total of 4577 differentially-expressed genes (DEGs were identified between the two floral stages. The Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that the DEGs in the “oxidation-reduction process”, “extracellular region”, “steroid biosynthesis”, “glycosphingolipid biosynthesis”, “plant hormone signal transduction” and “pentose and glucuronate interconversions” might be associated with flower development. A total of 103 and 92 unigenes exhibited sequence similarities to the known flower development and floral scent genes from other plants. Among these unigenes, five flower development and 19 floral scent unigenes exhibited at least four-fold differences in expression between the two stages. Our results provide abundant genetic resources for studying the flower blooming mechanisms and molecular breeding of J. sambac.

  4. Transcriptomic Analysis of Flower Blooming in Jasminum sambac through De Novo RNA Sequencing.

    Science.gov (United States)

    Li, Yong-Hua; Zhang, Wei; Li, Yong

    2015-06-10

    Flower blooming is a critical and complicated plant developmental process in flowering plants. However, insufficient information is available about the complex network that regulates flower blooming in Jasminum sambac. In this study, we used the RNA-Seq platform to analyze the molecular regulation of flower blooming in J. sambac by comparing the transcript profiles at two flower developmental stages: budding and blooming. A total of 4577 differentially-expressed genes (DEGs) were identified between the two floral stages. The Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that the DEGs in the "oxidation-reduction process", "extracellular region", "steroid biosynthesis", "glycosphingolipid biosynthesis", "plant hormone signal transduction" and "pentose and glucuronate interconversions" might be associated with flower development. A total of 103 and 92 unigenes exhibited sequence similarities to the known flower development and floral scent genes from other plants. Among these unigenes, five flower development and 19 floral scent unigenes exhibited at least four-fold differences in expression between the two stages. Our results provide abundant genetic resources for studying the flower blooming mechanisms and molecular breeding of J. sambac.

  5. MRUniNovo: an efficient tool for de novo peptide sequencing utilizing the hadoop distributed computing framework.

    Science.gov (United States)

    Li, Chuang; Chen, Tao; He, Qiang; Zhu, Yunping; Li, Kenli

    2016-12-19

    Tandem mass spectrometry-based de novo peptide sequencing is a complex and time-consuming process. The current algorithms for de novo peptide sequencing cannot rapidly and thoroughly process large mass spectrometry datasets. In this paper, we propose MRUniNovo, a novel tool for parallel de novo peptide sequencing. MRUniNovo parallelizes UniNovo based on the Hadoop compute platform. Our experimental results demonstrate that MRUniNovo significantly reduces the computation time of de novo peptide sequencing without sacrificing the correctness and accuracy of the results, and thus can process very large datasets that UniNovo cannot.

  6. Transcriptome de novo assembly sequencing and analysis of the toxic dinoflagellate Alexandrium catenella using the Illumina platform.

    Science.gov (United States)

    Zhang, Shu; Sui, Zhenghong; Chang, Lianpeng; Kang, Kyoungho; Ma, Jinhua; Kong, Fanna; Zhou, Wei; Wang, Jinguo; Guo, Liliang; Geng, Huili; Zhong, Jie; Ma, Qingxia

    2014-03-10

    In this article, high-throughput de novo transcriptomic sequencing was performed in Alexandrium catenella, which provided the first view of the gene repertoire in this dinoflagellate based on next-generation sequencing (NGS) technologies. A total of 118,304 unigenes were identified with an average length of 673bp (base pair). Of these unigenes, 77,936 (65.9%) were annotated with known proteins based on sequence similarities, among which 24,149 and 22,956 unigenes were assigned to gene ontology categories (GO) and clusters of orthologous groups (COGs), respectively. Furthermore, 16,467 unigenes were mapped onto 322 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). We also detected 1143 simple sequence repeats (SSRs), in which the tri-nucleotide repeat motif (69.3%) was the most abundant. The genetic facts and significance derived from the transcriptome dataset were suggested and discussed. All four core nucleosomal histones and linker histones were detected, in addition to the unigenes involved in histone modifications.190 unigenes were identified as being involved in the endocytosis pathway, and clathrin-dependent endocytosis was suggested to play a role in the heterotrophy of A. catenella. A conserved 22-nt spliced leader (SL) was identified in 21 unigenes which suggested the existence of trans-splicing processing of mRNA in A. catenella.

  7. Mass Spectrometry Analysis Coupled with de novo Sequencing Reveals Amino Acid Substitutions in Nucleocapsid Protein from Influenza A Virus

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    Zijian Li

    2014-02-01

    Full Text Available Amino acid substitutions in influenza A virus are the main reasons for both antigenic shift and virulence change, which result from non-synonymous mutations in the viral genome. Nucleocapsid protein (NP, one of the major structural proteins of influenza virus, is responsible for regulation of viral RNA synthesis and replication. In this report we used LC-MS/MS to analyze tryptic digestion of nucleocapsid protein of influenza virus (A/Puerto Rico/8/1934 H1N1, which was isolated and purified by SDS poly-acrylamide gel electrophoresis. Thus, LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three substituted amino acid residues R452K, T423A and N430T in two tryptic peptides. The obtained results provided experimental evidence that amino acid substitutions resulted from non-synonymous gene mutations could be directly characterized by mass spectrometry in proteins of RNA viruses such as influenza A virus.

  8. De novo transcriptome sequencing analysis and comparison of differentially expressed genes (DEGs in Macrobrachium rosenbergii in China.

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    Hai Nguyen Thanh

    Full Text Available Giant freshwater prawn (GFP; Macrobrachium rosenbergii is an exotic species that was introduced into China in 1976 and thereafter it became a major species in freshwater aquaculture. However the gene discovery in this species has been limited to small-scale data collection in China. We used the next generation sequencing technology for the experiment; the transcriptome was sequenced of samples of hepatopancreas organ in individuals from 4 GFP groups (A1, A2, B1 and B2. De novo transcriptome sequencing generated 66,953 isogenes. Using BLASTX to search the Non-redundant (NR, Search Tool for the Retrieval of Interacting Genes (STRING, and Kyoto Encyclopedia of Genes and Genome (KEGG databases; 21,224 unigenes were annotated, 9,552 matched unigenes with the Gene Ontology (GO classification; 5,782 matched unigenes in 25 categories of Clusters of Orthologous Groups of proteins (COG and 20,859 unigenes were consequently assigned to 312 KEGG pathways. Between the A and B groups 147 differentially expressed genes (DEGs were identified; between the A1 and A2 groups 6,860 DEGs were identified and between the B1 and B2 groups 5,229 DEGs were identified. After enrichment, the A and B groups identified 38 DEGs, but none of them were significantly enriched. The A1 and A2 groups identified 21,856 DEGs in three main categories based on functional groups: biological process, cellular_component and molecular function and the KEGG pathway defined 2,459 genes had a KEGG Ortholog-ID (KO-ID and could be categorized into 251 pathways, of those, 9 pathways were significantly enriched. The B1 and B2 groups identified 5,940 DEGs in three main categories based on functional groups: biological process, cellular_component and molecular function, and the KEGG pathway defined 1,543 genes had a KO-ID and could be categorized into 240 pathways, of those, 2 pathways were significantly enriched. We investigated 99 queries (GO which related to growth of GFP in 4 groups. After

  9. De novo transcriptome sequencing analysis and comparison of differentially expressed genes (DEGs) in Macrobrachium rosenbergii in China.

    Science.gov (United States)

    Nguyen Thanh, Hai; Zhao, Liangjie; Liu, Qigen

    2014-01-01

    Giant freshwater prawn (GFP; Macrobrachium rosenbergii) is an exotic species that was introduced into China in 1976 and thereafter it became a major species in freshwater aquaculture. However the gene discovery in this species has been limited to small-scale data collection in China. We used the next generation sequencing technology for the experiment; the transcriptome was sequenced of samples of hepatopancreas organ in individuals from 4 GFP groups (A1, A2, B1 and B2). De novo transcriptome sequencing generated 66,953 isogenes. Using BLASTX to search the Non-redundant (NR), Search Tool for the Retrieval of Interacting Genes (STRING), and Kyoto Encyclopedia of Genes and Genome (KEGG) databases; 21,224 unigenes were annotated, 9,552 matched unigenes with the Gene Ontology (GO) classification; 5,782 matched unigenes in 25 categories of Clusters of Orthologous Groups of proteins (COG) and 20,859 unigenes were consequently assigned to 312 KEGG pathways. Between the A and B groups 147 differentially expressed genes (DEGs) were identified; between the A1 and A2 groups 6,860 DEGs were identified and between the B1 and B2 groups 5,229 DEGs were identified. After enrichment, the A and B groups identified 38 DEGs, but none of them were significantly enriched. The A1 and A2 groups identified 21,856 DEGs in three main categories based on functional groups: biological process, cellular_component and molecular function and the KEGG pathway defined 2,459 genes had a KEGG Ortholog-ID (KO-ID) and could be categorized into 251 pathways, of those, 9 pathways were significantly enriched. The B1 and B2 groups identified 5,940 DEGs in three main categories based on functional groups: biological process, cellular_component and molecular function, and the KEGG pathway defined 1,543 genes had a KO-ID and could be categorized into 240 pathways, of those, 2 pathways were significantly enriched. We investigated 99 queries (GO) which related to growth of GFP in 4 groups. After enrichment we

  10. Comparative analysis of two phenologically divergent populations of the pine processionary moth (Thaumetopoea pityocampa) by de novo transcriptome sequencing.

    Science.gov (United States)

    Gschloessl, Bernhard; Vogel, Heiko; Burban, Christian; Heckel, David; Streiff, Réjane; Kerdelhué, Carole

    2014-03-01

    The pine processionary moth Thaumetopoea pityocampa is a Mediterranean lepidopteran defoliator that experiences a rapid range expansion towards higher latitudes and altitudes due to the current climate warming. Its phenology - the time of sexual reproduction - is certainly a key trait for the local adaptation of the processionary moth to climatic conditions. Moreover, an exceptional case of allochronic differentiation was discovered ca. 15 years ago in this species. A population with a shifted phenology (the summer population, SP) co-exists near Leiria, Portugal, with a population following the classical cycle (the winter population, WP). The existence of this population is an outstanding opportunity to decipher the genetic bases of phenology. No genomic resources were so far available for T. pityocampa. We developed a high-throughput sequencing approach to build a first reference transcriptome, and to proceed with comparative analyses of the sympatric SP and WP. We pooled RNA extracted from whole individuals of various developmental stages, and performed a transcriptome characterisation for both populations combining Roche 454-FLX and traditional Sanger data. The obtained sequences were clustered into ca. 12,000 transcripts corresponding to 9265 unigenes. The mean transcript coverage was 21.9 reads per bp. Almost 70% of the de novo assembled transcripts displayed significant similarity to previously published proteins and around 50% of the transcripts contained a full-length coding region. Comparative analyses of the population transcriptomes allowed to investigate genes specifically expressed in one of the studied populations only, and to identify the most divergent homologous SP/WP transcripts. The most divergent pairs of transcripts did not correspond to obvious phenology-related candidate genes, and 43% could not be functionally annotated. This study provides the first comprehensive genome-wide resource for the target species T. pityocampa. Many of the

  11. Transcriptome analysis of colored calla lily (Zantedeschia rehmannii Engl.) by Illumina sequencing: de novo assembly, annotation and EST-SSR marker development.

    Science.gov (United States)

    Wei, Zunzheng; Sun, Zhenzhen; Cui, Binbin; Zhang, Qixiang; Xiong, Min; Wang, Xian; Zhou, Di

    2016-01-01

    Colored calla lily is the short name for the species or hybrids in section Aestivae of genus Zantedeschia. It is currently one of the most popular flower plants in the world due to its beautiful flower spathe and long postharvest life. However, little genomic information and few molecular markers are available for its genetic improvement. Here, de novo transcriptome sequencing was performed to produce large transcript sequences for Z. rehmannii cv. 'Rehmannii' using an Illumina HiSeq 2000 instrument. More than 59.9 million cDNA sequence reads were obtained and assembled into 39,298 unigenes with an average length of 1,038 bp. Among these, 21,077 unigenes showed significant similarity to protein sequences in the non-redundant protein database (Nr) and in the Swiss-Prot, Gene Ontology (GO), Cluster of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Moreover, a total of 117 unique transcripts were then defined that might regulate the flower spathe development of colored calla lily. Additionally, 9,933 simple sequence repeats (SSRs) and 7,162 single nucleotide polymorphisms (SNPs) were identified as putative molecular markers. High-quality primers for 200 SSR loci were designed and selected, of which 58 amplified reproducible amplicons were polymorphic among 21 accessions of colored calla lily. The sequence information and molecular markers in the present study will provide valuable resources for genetic diversity analysis, germplasm characterization and marker-assisted selection in the genus Zantedeschia.

  12. De Novo RNA Sequencing and Transcriptome Analysis of Monascus purpureus and Analysis of Key Genes Involved in Monacolin K Biosynthesis

    Science.gov (United States)

    Zhang, Chan; Liang, Jian; Yang, Le; Sun, Baoguo; Wang, Chengtao

    2017-01-01

    Monascus purpureus is an important medicinal and edible microbial resource. To facilitate biological, biochemical, and molecular research on medicinal components of M. purpureus, we investigated the M. purpureus transcriptome by RNA sequencing (RNA-seq). An RNA-seq library was created using RNA extracted from a mixed sample of M. purpureus expressing different levels of monacolin K output. In total 29,713 unigenes were assembled from more than 60 million high-quality short reads. A BLAST search revealed hits for 21,331 unigenes in at least one of the protein or nucleotide databases used in this study. The 22,365 unigenes were categorized into 48 functional groups based on Gene Ontology classification. Owing to the economic and medicinal importance of M. purpureus, most studies on this organism have focused on the pharmacological activity of chemical components and the molecular function of genes involved in their biogenesis. In this study, we performed quantitative real-time PCR to detect the expression of genes related to monacolin K (mokA-mokI) at different phases (2, 5, 8, and 12 days) of M. purpureus M1 and M1-36. Our study found that mokF modulates monacolin K biogenesis in M. purpureus. Nine genes were suggested to be associated with the monacolin K biosynthesis. Studies on these genes could provide useful information on secondary metabolic processes in M. purpureus. These results indicate a detailed resource through genetic engineering of monacolin K biosynthesis in M. purpureus and related species. PMID:28114365

  13. De novo sequencing and analysis of the transcriptome of Panax ginseng in the leaf-expansion period.

    Science.gov (United States)

    Liu, Shichao; Wang, Siming; Liu, Meichen; Yang, Fei; Zhang, Hui; Liu, Shiyang; Wang, Qun; Zhao, Yu

    2016-08-01

    Panax ginseng, a traditional Chinese medicine, is used worldwide for its variety of health benefits and its treatment efficacy. However, it is difficult to cultivate due to its vulnerability to environmental stresses. The present study provided the first report, to the best of our knowledge, of transcriptome analysis of ginseng at the leaf‑expansion stage. Using the Illumina sequencing platform, >40,000,000 high‑quality paired‑end reads were obtained and assembled into 100,533 unique sequences. When the sequences were searched against the publicly available National Center for Biotechnology Information protein database using The Basic Local Alignment Search Tool, 61,599 sequences exhibited similarity to known proteins. Functional annotation and classification, including use of the Gene Ontology, Clusters of Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes databases, revealed that the activated genes in ginseng were predominantly ribonuclease‑like storage genes, environmental stress genes, pathogenesis-related genes and other antioxidant genes. A number of candidate genes in environmental stress‑associated pathways were also identified. These novel data provide useful information on the growth and development stages of ginseng, and serve as an important public information platform for further understanding of the molecular mechanisms and functional genomics of ginseng.

  14. De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress.

    Science.gov (United States)

    Xu, Peng; Liu, Zhangwei; Fan, Xinqi; Gao, Jin; Zhang, Xia; Zhang, Xianggui; Shen, Xinlian

    2013-08-01

    Salinity stress is one of the most serious factors that impede the growth and development of various crops. Wild Gossypium species, which are remarkably tolerant to salt water immersion, are valuable resources for understanding salt tolerance mechanisms of Gossypium and improving salinity resistance in upland cotton. To generate a broad survey of genes with altered expression during various stages of salt stress, a mixed RNA sample was prepared from the roots and leaves of Gossypium aridum plants subjected to salt stress. The transcripts were sequenced using the Illumina sequencing platform. After cleaning and quality checks, approximately 41.5 million clean reads were obtained. Finally, these reads were eventually assembled into 98,989 unigenes with a mean size of 452 bp. All unigenes were compared to known cluster of orthologous groups (COG) sequences to predict and classify the possible functions of these genes, which were classified into at least 25 molecular families. Variations in gene expression were then examined after exposing the plants to 200 mM NaCl for 3, 12, 72 or 144 h. Sequencing depths of approximately six million raw tags were achieved for each of the five stages of salt stress. There were 2634 (1513 up-regulated/1121 down-regulated), 2449 (1586 up-regulated/863 down-regulated), 2271 (946 up-regulated/1325 down-regulated) and 3352 (933 up-regulated/2419 down-regulated) genes that were differentially expressed after exposure to NaCl for 3, 12, 72 and 144 h, respectively. Digital gene expression analysis indicated that pathways involved in "transport", "response to hormone stimulus" and "signaling" play important roles during salt stress, while genes involved in "protein kinase activity" and "transporter activity" undergo major changes in expression during early and later stages of salt stress, respectively.

  15. De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis

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    Lui Edmund MK

    2010-04-01

    Full Text Available Abstract Background American ginseng (Panax quinquefolius L. is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway. Results In this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8% unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450 and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay. Conclusions We demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the

  16. Novor: real-time peptide de novo sequencing software.

    Science.gov (United States)

    Ma, Bin

    2015-11-01

    De novo sequencing software has been widely used in proteomics to sequence new peptides from tandem mass spectrometry data. This study presents a new software tool, Novor, to greatly improve both the speed and accuracy of today's peptide de novo sequencing analyses. To improve the accuracy, Novor's scoring functions are based on two large decision trees built from a peptide spectral library with more than 300,000 spectra with machine learning. Important knowledge about peptide fragmentation is extracted automatically from the library and incorporated into the scoring functions. The decision tree model also enables efficient score calculation and contributes to the speed improvement. To further improve the speed, a two-stage algorithmic approach, namely dynamic programming and refinement, is used. The software program was also carefully optimized. On the testing datasets, Novor sequenced 7%-37% more correct residues than the state-of-the-art de novo sequencing tool, PEAKS, while being an order of magnitude faster. Novor can de novo sequence more than 300 MS/MS spectra per second on a laptop computer. The speed surpasses the acquisition speed of today's mass spectrometer and, therefore, opens a new possibility to de novo sequence in real time while the spectrometer is acquiring the spectral data. Graphical Abstract ᅟ.

  17. Novor: Real-Time Peptide de Novo Sequencing Software

    Science.gov (United States)

    Ma, Bin

    2015-11-01

    De novo sequencing software has been widely used in proteomics to sequence new peptides from tandem mass spectrometry data. This study presents a new software tool, Novor, to greatly improve both the speed and accuracy of today's peptide de novo sequencing analyses. To improve the accuracy, Novor's scoring functions are based on two large decision trees built from a peptide spectral library with more than 300,000 spectra with machine learning. Important knowledge about peptide fragmentation is extracted automatically from the library and incorporated into the scoring functions. The decision tree model also enables efficient score calculation and contributes to the speed improvement. To further improve the speed, a two-stage algorithmic approach, namely dynamic programming and refinement, is used. The software program was also carefully optimized. On the testing datasets, Novor sequenced 7%-37% more correct residues than the state-of-the-art de novo sequencing tool, PEAKS, while being an order of magnitude faster. Novor can de novo sequence more than 300 MS/MS spectra per second on a laptop computer. The speed surpasses the acquisition speed of today's mass spectrometer and, therefore, opens a new possibility to de novo sequence in real time while the spectrometer is acquiring the spectral data.

  18. De novo sequencing and comparative analysis of three red algal species of Family Solieriaceae to discover putative genes associated with carrageenan biosysthesis

    Institute of Scientific and Technical Information of China (English)

    SONG Lipu; WANG Xumin; YU Jun; WU Shuangxiu; SUN Jing; WANG Liang; LIU Tao; CHI Shan; LIU Cui; LI Xingang; YIN Jinlong

    2014-01-01

    Betaphycus gelatinus, Kappaphycus alvarezii and Eucheuma denticulatum of Family Solieriaceae, Order Gi-gartinales, Class Rhodophyceae are three important carrageenan-producing red algal species, which pro-duce different types of carrageenans, beta (β)-carrageenan, kappa (κ)-carrageenan and iota (ι)-carrageenan. So far the carrageenan biosynthesis pathway is not fully understood and few information is about the So-lieriaceae genome and transcriptome sequence. Here, we performed the de novo transcriptome sequencing, assembly, functional annotation and comparative analysis of these three commercial-valuable species using an Illumina short-sequencing platform Hiseq 2000 and bioinformatic software. Furthermore, we compared the different expression of some unigenes involved in some pathways relevant to carrageenan biosynthe-sis. We finally found 861 different expressed KEGG orthologs which contained a glycolysis/gluconeogenesis pathway (21 orthologs), carbon fixation in photosynthetic organisms (16 orthologs), galactose metabolism (5 orthologs), and fructose and mannose metabolism (9 orthologs) which are parts of the carbohydrate me-tabolism. We also found 8 different expressed KEGG orthologs for sulfur metabolism which might be impor-tantly related to biosynthesis of different types of carrageenans. The results presented in this study provided valuable resources for functional genomics annotation and investigation of mechanisms underlying the biosynthesis of carrageenan in Family Solieriaceae.

  19. Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery

    Science.gov (United States)

    Sen, K. Ilker; Tang, Wilfred H.; Nayak, Shruti; Kil, Yong J.; Bern, Marshall; Ozoglu, Berk; Ueberheide, Beatrix; Davis, Darryl; Becker, Christopher

    2017-01-01

    Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests.

  20. Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery

    Science.gov (United States)

    Sen, K. Ilker; Tang, Wilfred H.; Nayak, Shruti; Kil, Yong J.; Bern, Marshall; Ozoglu, Berk; Ueberheide, Beatrix; Davis, Darryl; Becker, Christopher

    2017-05-01

    Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests.

  1. Sequencing and de novo analysis of the hemocytes transcriptome in Litopenaeus vannamei response to white spot syndrome virus infection.

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    Shuxia Xue

    Full Text Available BACKGROUND: White spot syndrome virus (WSSV is a causative pathogen found in most shrimp farming areas of the world and causes large economic losses to the shrimp aquaculture. The mechanism underlying the molecular pathogenesis of the highly virulent WSSV remains unknown. To better understand the virus-host interactions at the molecular level, the transcriptome profiles in hemocytes of unchallenged and WSSV-challenged shrimp (Litopenaeus vannamei were compared using a short-read deep sequencing method (Illumina. RESULTS: RNA-seq analysis generated more than 25.81 million clean pair end (PE reads, which were assembled into 52,073 unigenes (mean size = 520 bp. Based on sequence similarity searches, 23,568 (45.3% genes were identified, among which 6,562 and 7,822 unigenes were assigned to gene ontology (GO categories and clusters of orthologous groups (COG, respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG mapped 14,941 (63.4% unigenes to 240 KEGG pathways. Among all the annotated unigenes, 1,179 were associated with immune-related genes. Digital gene expression (DGE analysis revealed that the host transcriptome profile was slightly changed in the early infection (5 hours post injection of the virus, while large transcriptional differences were identified in the late infection (48 hpi of WSSV. The differentially expressed genes mainly involved in pattern recognition genes and some immune response factors. The results indicated that antiviral immune mechanisms were probably involved in the recognition of pathogen-associated molecular patterns. CONCLUSIONS: This study provided a global survey of host gene activities against virus infection in a non-model organism, pacific white shrimp. Results can contribute to the in-depth study of candidate genes in white shrimp, and help to improve the current understanding of host-pathogen interactions.

  2. Transcriptome sequencing and de novo analysis of cytoplasmic male sterility and maintenance in JA-CMS cotton.

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    Peng Yang

    Full Text Available Cytoplasmic male sterility (CMS is the failure to produce functional pollen, which is inherited maternally. And it is known that anther development is modulated through complicated interactions between nuclear and mitochondrial genes in sporophytic and gametophytic tissues. However, an unbiased transcriptome sequencing analysis of CMS in cotton is currently lacking in the literature. This study compared differentially expressed (DE genes of floral buds at the sporogenous cells stage (SS and microsporocyte stage (MS (the two most important stages for pollen abortion in JA-CMS between JA-CMS and its fertile maintainer line JB cotton plants, using the Illumina HiSeq 2000 sequencing platform. A total of 709 (1.8% DE genes including 293 up-regulated and 416 down-regulated genes were identified in JA-CMS line comparing with its maintainer line at the SS stage, and 644 (1.6% DE genes with 263 up-regulated and 381 down-regulated genes were detected at the MS stage. By comparing the two stages in the same material, there were 8 up-regulated and 9 down-regulated DE genes in JA-CMS line and 29 up-regulated and 9 down-regulated DE genes in JB maintainer line at the MS stage. Quantitative RT-PCR was used to validate 7 randomly selected DE genes. Bioinformatics analysis revealed that genes involved in reduction-oxidation reactions and alpha-linolenic acid metabolism were down-regulated, while genes pertaining to photosynthesis and flavonoid biosynthesis were up-regulated in JA-CMS floral buds compared with their JB counterparts at the SS and/or MS stages. All these four biological processes play important roles in reactive oxygen species (ROS homeostasis, which may be an important factor contributing to the sterile trait of JA-CMS. Further experiments are warranted to elucidate molecular mechanisms of these genes that lead to CMS.

  3. Transcriptome sequencing and de novo analysis of cytoplasmic male sterility and maintenance in JA-CMS cotton.

    Science.gov (United States)

    Yang, Peng; Han, Jinfeng; Huang, Jinling

    2014-01-01

    Cytoplasmic male sterility (CMS) is the failure to produce functional pollen, which is inherited maternally. And it is known that anther development is modulated through complicated interactions between nuclear and mitochondrial genes in sporophytic and gametophytic tissues. However, an unbiased transcriptome sequencing analysis of CMS in cotton is currently lacking in the literature. This study compared differentially expressed (DE) genes of floral buds at the sporogenous cells stage (SS) and microsporocyte stage (MS) (the two most important stages for pollen abortion in JA-CMS) between JA-CMS and its fertile maintainer line JB cotton plants, using the Illumina HiSeq 2000 sequencing platform. A total of 709 (1.8%) DE genes including 293 up-regulated and 416 down-regulated genes were identified in JA-CMS line comparing with its maintainer line at the SS stage, and 644 (1.6%) DE genes with 263 up-regulated and 381 down-regulated genes were detected at the MS stage. By comparing the two stages in the same material, there were 8 up-regulated and 9 down-regulated DE genes in JA-CMS line and 29 up-regulated and 9 down-regulated DE genes in JB maintainer line at the MS stage. Quantitative RT-PCR was used to validate 7 randomly selected DE genes. Bioinformatics analysis revealed that genes involved in reduction-oxidation reactions and alpha-linolenic acid metabolism were down-regulated, while genes pertaining to photosynthesis and flavonoid biosynthesis were up-regulated in JA-CMS floral buds compared with their JB counterparts at the SS and/or MS stages. All these four biological processes play important roles in reactive oxygen species (ROS) homeostasis, which may be an important factor contributing to the sterile trait of JA-CMS. Further experiments are warranted to elucidate molecular mechanisms of these genes that lead to CMS.

  4. De novo DNA sequence driven bulk segregant analysis of Water Use Efficiency (WUE) in potato without prior knowledge of molecular markers

    DEFF Research Database (Denmark)

    Kaminski, Kacper Piotr; Sønderkær, Mads; Sørensen, Kirsten Kørup;

    2012-01-01

    concentration on leaf surface as well as temperature were analyzed and their consequences on water use efficiency were evaluated during the climate chamber experiments in 2010 and 2011. We have previously participated in development of a de novo sequence-based alternative to positional cloning (SHOREMap)2. Here....... Four bulks (two of high and two of low WUE) and the parents were subsequently sequenced to ~30x coverage. Non-random distribution of sequence read-based polymorphisms between the WUE pools and the parental pool delimited highly resolved regions on several chromosomes. Within these regions candidate...

  5. De novo sequencing and analysis of the lily pollen transcriptome: an open access data source for an orphan plant species.

    Science.gov (United States)

    Lang, Veronika; Usadel, Björn; Obermeyer, Gerhard

    2015-01-01

    Pollen grains of Lilium longiflorum are a long-established model system for pollen germination and tube tip growth. Due to their size, protein content and almost synchronous germination in synthetic media, they provide a simple system for physiological measurements as well as sufficient material for biochemical studies like protein purifications, enzyme assays, organelle isolation or determination of metabolites during germination and pollen tube elongation. Despite recent progresses in molecular biology techniques, sequence information of expressed proteins or transcripts in lily pollen is still scarce. Using a next generation sequencing strategy (RNAseq), the lily pollen transcriptome was investigated resulting in more than 50 million high quality reads with a length of 90 base pairs. Sequenced transcripts were assembled and annotated, and finally visualized with MAPMAN software tools and compared with other RNAseq or genome data including Arabidopsis pollen, Lilium vegetative tissues and the Amborella trichopoda genome. All lily pollen sequence data are provided as open access files with suitable tools to search sequences of interest.

  6. De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue-green alga Aphanizomenon flos-aquae.

    Science.gov (United States)

    Rinalducci, Sara; Roepstorff, Peter; Zolla, Lello

    2009-04-01

    In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos-aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) and MALDI-TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both alpha- and beta-phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI- and ESI-MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species. Copyright (c) 2009 John Wiley & Sons, Ltd.

  7. De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley

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    Scholz Uwe

    2009-11-01

    Full Text Available Abstract Background De novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L. is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable. Results To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of ~50 kb (N80 ~31 kb, N90 ~21 kb and a Q40 of 94%. For ~80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes. By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies. Conclusion The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome.

  8. De novo Transcriptome Analysis in Perennial Ryegrass

    DEFF Research Database (Denmark)

    Farrell, Jacqueline Danielle; Byrne, Stephen; Asp, Torben

    selection will be the availability of a reference genome, and efforts are underway within our group to deliver this. An important step in de novo assembly will be defining the gene set, and the availability of transcriptome sequencing data will greatly aid gene prediction and validation, and the development...... of functional markers for improved ryegrass breeding. Therefore, the goal of this study is to analyze a de novo assembly of the perennial ryegrass transcriptome from the same inbred genotype being used for de novo genome assembly. Furthermore, we also conducted de novo transcriptome assembly with other......Perennial ryegrass (Lolium perenne L.) is an important grass species for both forage and amenity purposes for temperate regions worldwide. It is envisaged that breeding efforts may be enhanced with the assistance of new breeding technologies such as genomic selection. A major step towards genomic...

  9. De novo Transcriptome Sequencing and Development of Abscission Zone-Specific Microarray as a New Molecular Tool for Analysis of Tomato Organ Abscission

    Science.gov (United States)

    Sundaresan, Srivignesh; Philosoph-Hadas, Sonia; Riov, Joseph; Mugasimangalam, Raja; Kuravadi, Nagesh A.; Kochanek, Bettina; Salim, Shoshana; Tucker, Mark L.; Meir, Shimon

    2016-01-01

    Abscission of flower pedicels and leaf petioles of tomato (Solanum lycopersicum) can be induced by flower removal or leaf deblading, respectively, which leads to auxin depletion, resulting in increased sensitivity of the abscission zone (AZ) to ethylene. However, the molecular mechanisms that drive the acquisition of abscission competence and its modulation by auxin gradients are not yet known. We used RNA-Sequencing (RNA-Seq) to obtain a comprehensive transcriptome of tomato flower AZ (FAZ) and leaf AZ (LAZ) during abscission. RNA-Seq was performed on a pool of total RNA extracted from tomato FAZ and LAZ, at different abscission stages, followed by de novo assembly. The assembled clusters contained transcripts that are already known in the Solanaceae (SOL) genomics and NCBI databases, and over 8823 identified novel tomato transcripts of varying sizes. An AZ-specific microarray, encompassing the novel transcripts identified in this study and all known transcripts from the SOL genomics and NCBI databases, was constructed to study the abscission process. Multiple probes for longer genes and key AZ-specific genes, including antisense probes for all transcripts, make this array a unique tool for studying abscission with a comprehensive set of transcripts, and for mining for naturally occurring antisense transcripts. We focused on comparing the global transcriptomes generated from the FAZ and the LAZ to establish the divergences and similarities in their transcriptional networks, and particularly to characterize the processes and transcriptional regulators enriched in gene clusters that are differentially regulated in these two AZs. This study is the first attempt to analyze the global gene expression in different AZs in tomato by combining the RNA-Seq technique with oligonucleotide microarrays. Our AZ-specific microarray chip provides a cost-effective approach for expression profiling and robust analysis of multiple samples in a rapid succession. PMID:26834766

  10. De novo transcriptome sequencing and development of abscission zone-specific microarray as a new molecular tool for analysis of tomato organ abscission

    Directory of Open Access Journals (Sweden)

    Srivignesh eSundaresan

    2016-01-01

    Full Text Available Abscission of flower pedicels and leaf petioles of tomato (Solanum lycopersicum can be induced by flower removal or leaf deblading, respectively, leading to auxin depletion, which results in increased sensitivity of the abscission zone (AZ to ethylene. However, the molecular mechanisms that drive the acquisition of abscission competence and its modulation by auxin gradients are not yet known. We used RNA-Sequencing (RNA-Seq to obtain the comprehensive transcriptome of tomato flower AZ (FAZ and leaf AZ (LAZ during abscission. RNA-Seq was performed on a pool of total RNA extracted from different abscission stages of tomato FAZ and LAZ, followed by de novo assembly. The assembled clusters contained transcripts that are already known in Solanaceae (SOL genomics database and NCBI databases, and over 8,823 identified novel tomato transcripts of varying sizes. An AZ-specific microarray, encompassing these novel transcripts identified in this study and all known transcripts from the SOL genomics and NCBI databases, was constructed to study the abscission process. Multiple probes for longer genes and key AZ-specific genes, including antisense probes for all transcripts, make this array a unique tool for studying abscission with a comprehensive set of transcripts, and for mining for naturally occurring antisense transcripts. We focused on comparing the global transcriptomes generated from the FAZ and the LAZ to establish the divergences and similarities in their transcriptional networks, and particularly to characterize the processes and transcriptional regulators enriched in gene clusters that are differentially regulated in these two AZs. This study is the first attempt to analyze the global gene expression in different AZs in tomato by combining the RNA-Seq technique with oligonucleotide microarrays. Our AZ-specific microarray chip provides a cost-effective approach for expression profiling and robust analysis of multiple samples in a rapid succession.

  11. De novo Sequencing and Transcriptome Analysis Reveal Key Genes Regulating Steroid Metabolism in Leaves, Roots, Adventitious Roots and Calli of Periploca sepium Bunge

    Directory of Open Access Journals (Sweden)

    Xinglin Li

    2017-04-01

    Full Text Available Periploca sepium Bunge is a traditional medicinal plant, whose root bark is important for Chinese herbal medicine. Its major bioactive compounds are C21 steroids and periplocin, a kind of cardiac glycoside, which are derived from the steroid synthesis pathway. However, research on P. sepium genome or transcriptomes and their related genes has been lacking for a long time. In this study we estimated this species nuclear genome size at 170 Mb (using flow cytometry. Then, RNA sequencing of four different tissue samples of P. sepium (leaves, roots, adventitious roots, and calli was done using the sequencing platform Illumina/Solexa Hiseq 2,500. After de novo assembly and quantitative assessment, 90,375 all-transcripts and 71,629 all-unigenes were finally generated. Annotation efforts that used a number of public databases resulted in detailed annotation information for the transcripts. In addition, differentially expressed genes (DEGs were identified by using digital gene profiling based on the reads per kilobase of transcript per million reads mapped (RPKM values. Compared with the leaf samples (L, up-regulated genes and down-regulated genes were eventually obtained. To deepen our understanding of these DEGs, we performed two enrichment analyses: gene ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG. Here, the analysis focused upon the expression characteristics of those genes involved in the terpene metabolic pathway and the steroid biosynthesis pathway, to better elucidate the molecular mechanism of bioactive steroid synthesis in P. sepium. The bioinformatics analysis enabled us to find many genes that are involved in bioactive steroid biosynthesis. These genes encoded acetyl-CoA acetyltransferase (ACAT, HMG-CoA synthase (HMGS, HMG-CoA reductase (HMGR, mevalonate kinase (MK, phosphomevalonate kinase (PMK, mevalonate diphosphate decarboxylase (MDD, isopentenylpyrophosphate isomerase (IPPI, farnesyl pyrophosphate synthase (FPS

  12. Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

    Science.gov (United States)

    Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

    2013-01-01

    Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

  13. Proteomics of Soil and Sediment: Protein Identification by De Novo Sequencing of Mass Spectra Complements Traditional Database Searching

    Science.gov (United States)

    Miller, S.; Rizzo, A. I.; Waldbauer, J.

    2015-12-01

    Proteomics has the potential to elucidate the metabolic pathways and taxa responsible for in situ biogeochemical transformations. However, low rates of protein identification from high resolution mass spectra have been a barrier to the development of proteomics in complex environmental samples. Much of the difficulty lies in the computational challenge of linking mass spectra to their corresponding proteins. Traditional database search methods for matching peptide sequences to mass spectra are often inadequate due to the complexity of environmental proteomes and the large database search space, as we demonstrate with soil and sediment proteomes generated via a range of extraction methods. One alternative to traditional database searching is de novo sequencing, which identifies peptide sequences without the need for a database. BLAST can then be used to match de novo sequences to similar genetic sequences. Assigning confidence to putative identifications has been one hurdle for the implementation of de novo sequencing. We found that accurate de novo sequences can be screened by quality score and length. Screening criteria are verified by comparing the results of de novo sequencing and traditional database searching for well-characterized proteomes from simple biological systems. The BLAST hits of screened sequences are interrogated for taxonomic and functional information. We applied de novo sequencing to organic topsoil and marine sediment proteomes. Peak-rich proteomes, which can result from various extraction techniques, yield thousands of high-confidence protein identifications, an improvement over previous proteomic studies of soil and sediment. User-friendly software tools for de novo metaproteomics analysis have been developed. This "De Novo Analysis" Pipeline is also a faster method of data analysis than constructing a tailored sequence database for traditional database searching.

  14. iAssembler: a package for de novo assembly of Roche-454/Sanger transcriptome sequences

    Directory of Open Access Journals (Sweden)

    Zheng Yi

    2011-11-01

    Full Text Available Abstract Background Expressed Sequence Tags (ESTs have played significant roles in gene discovery and gene functional analysis, especially for non-model organisms. For organisms with no full genome sequences available, ESTs are normally assembled into longer consensus sequences for further downstream analysis. However current de novo EST assembly programs often generate large number of assembly errors that will negatively affect the downstream analysis. In order to generate more accurate consensus sequences from ESTs, tools are needed to reduce or eliminate errors from de novo assemblies. Results We present iAssembler, a pipeline that can assemble large-scale ESTs into consensus sequences with significantly higher accuracy than current existing assemblers. iAssembler employs MIRA and CAP3 assemblers to generate initial assemblies, followed by identifying and correcting two common types of transcriptome assembly errors: 1 ESTs from different transcripts (mainly alternatively spliced transcripts or paralogs are incorrectly assembled into same contigs; and 2 ESTs from same transcripts fail to be assembled together. iAssembler can be used to assemble ESTs generated using the traditional Sanger method and/or the Roche-454 massive parallel pyrosequencing technology. Conclusion We compared performances of iAssembler and several other de novo EST assembly programs using both Roche-454 and Sanger EST datasets. It demonstrated that iAssembler generated significantly more accurate consensus sequences than other assembly programs.

  15. Transcriptome sequencing and de novo analysis of a cytoplasmic male sterile line and its near-isogenic restorer line in chili pepper (Capsicum annuum L..

    Directory of Open Access Journals (Sweden)

    Chen Liu

    Full Text Available BACKGROUND: The use of cytoplasmic male sterility (CMS in F1 hybrid seed production of chili pepper is increasingly popular. However, the molecular mechanisms of cytoplasmic male sterility and fertility restoration remain poorly understood due to limited transcriptomic and genomic data. Therefore, we analyzed the difference between a CMS line 121A and its near-isogenic restorer line 121C in transcriptome level using next generation sequencing technology (NGS, aiming to find out critical genes and pathways associated with the male sterility. RESULTS: We generated approximately 53 million sequencing reads and assembled de novo, yielding 85,144 high quality unigenes with an average length of 643 bp. Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively. Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen. CONCLUSIONS: Our study profiled anther transcriptomes of a chili pepper CMS line and its restorer line. The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations.

  16. Transcriptome Sequencing and De Novo Analysis of a Cytoplasmic Male Sterile Line and Its Near-Isogenic Restorer Line in Chili Pepper (Capsicum annuum L.)

    Science.gov (United States)

    Wang, Ping-Yong; Fu, Nan; Shen, Huo-Lin

    2013-01-01

    Background The use of cytoplasmic male sterility (CMS) in F1 hybrid seed production of chili pepper is increasingly popular. However, the molecular mechanisms of cytoplasmic male sterility and fertility restoration remain poorly understood due to limited transcriptomic and genomic data. Therefore, we analyzed the difference between a CMS line 121A and its near-isogenic restorer line 121C in transcriptome level using next generation sequencing technology (NGS), aiming to find out critical genes and pathways associated with the male sterility. Results We generated approximately 53 million sequencing reads and assembled de novo, yielding 85,144 high quality unigenes with an average length of 643 bp. Among these unigenes, 27,191 were identified as putative homologs of annotated sequences in the public protein databases, 4,326 and 7,061 unigenes were found to be highly abundant in lines 121A and 121C, respectively. Many of the differentially expressed unigenes represent a set of potential candidate genes associated with the formation or abortion of pollen. Conclusions Our study profiled anther transcriptomes of a chili pepper CMS line and its restorer line. The results shed the lights on the occurrence and recovery of the disturbances in nuclear-mitochondrial interaction and provide clues for further investigations. PMID:23750245

  17. The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads

    DEFF Research Database (Denmark)

    Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo

    2012-01-01

    to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig....... A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K...... these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species....

  18. De novo ORFs in Drosophila are important to organismal fitness and evolved rapidly from previously non-coding sequences.

    Directory of Open Access Journals (Sweden)

    Josephine A Reinhardt

    Full Text Available How non-coding DNA gives rise to new protein-coding genes (de novo genes is not well understood. Recent work has revealed the origins and functions of a few de novo genes, but common principles governing the evolution or biological roles of these genes are unknown. To better define these principles, we performed a parallel analysis of the evolution and function of six putatively protein-coding de novo genes described in Drosophila melanogaster. Reconstruction of the transcriptional history of de novo genes shows that two de novo genes emerged from novel long non-coding RNAs that arose at least 5 MY prior to evolution of an open reading frame. In contrast, four other de novo genes evolved a translated open reading frame and transcription within the same evolutionary interval suggesting that nascent open reading frames (proto-ORFs, while not required, can contribute to the emergence of a new de novo gene. However, none of the genes arose from proto-ORFs that existed long before expression evolved. Sequence and structural evolution of de novo genes was rapid compared to nearby genes and the structural complexity of de novo genes steadily increases over evolutionary time. Despite the fact that these genes are transcribed at a higher level in males than females, and are most strongly expressed in testes, RNAi experiments show that most of these genes are essential in both sexes during metamorphosis. This lethality suggests that protein coding de novo genes in Drosophila quickly become functionally important.

  19. De novo sequencing-based transcriptome and digital gene expression analysis reveals insecticide resistance-relevant genes in Propylaea japonica (Thunberg (Coleoptea: Coccinellidae.

    Directory of Open Access Journals (Sweden)

    Liang-De Tang

    Full Text Available The ladybird Propylaea japonica (Thunberg is one of most important natural enemies of aphids in China. This species is threatened by the extensive use of insecticides but genomics-based information on the molecular mechanisms underlying insecticide resistance is limited. Hence, we analyzed the transcriptome and expression profile data of P. japonica in order to gain a deeper understanding of insecticide resistance in ladybirds. We performed de novo assembly of a transcriptome using Illumina's Solexa sequencing technology and short reads. A total of 27,243,552 reads were generated. These were assembled into 81,458 contigs and 33,647 unigenes (6,862 clusters and 26,785 singletons. Of the unigenes, 23,965 (71.22% have putative homologues in the non-redundant (nr protein database from NCBI, using BLASTX, with a cut-off E-value of 10(-5. We examined COG, GO and KEGG annotations to better understand the functions of these unigenes. Digital gene expression (DGE libraries showed differences in gene expression profiles between two insecticide resistant strains. When compared with an insecticide susceptible profile, a total of 4,692 genes were significantly up- or down- regulated in a moderately resistant strain. Among these genes, 125 putative insecticide resistance genes were identified. To confirm the DGE results, 16 selected genes were validated using quantitative real time PCR (qRT-PCR. This study is the first to report genetic information on P. japonica and has greatly enriched the sequence data for ladybirds. The large number of gene sequences produced from the transcriptome and DGE sequencing will greatly improve our understanding of this important insect, at the molecular level, and could contribute to the in-depth research into insecticide resistance mechanisms.

  20. De novo sequencing and transcriptome analysis of Pinellia ternata identify the candidate genes involved in the biosynthesis of benzoic acid and ephedrine

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    Zhang Guang Hui

    2016-08-01

    Full Text Available Background: The medicinal herb, Pinellia ternate, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable. Results: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeqTM 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternate, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR. Also, 14,468 simple sequence repeats (SSRs were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect. Conclusion: RNA-seq data was firstly used to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant.

  1. De Novo Peptide Sequencing: Deep Mining of High-Resolution Mass Spectrometry Data.

    Science.gov (United States)

    Islam, Mohammad Tawhidul; Mohamedali, Abidali; Fernandes, Criselda Santan; Baker, Mark S; Ranganathan, Shoba

    2017-01-01

    High resolution mass spectrometry has revolutionized proteomics over the past decade, resulting in tremendous amounts of data in the form of mass spectra, being generated in a relatively short span of time. The mining of this spectral data for analysis and interpretation though has lagged behind such that potentially valuable data is being overlooked because it does not fit into the mold of traditional database searching methodologies. Although the analysis of spectra by de novo sequences removes such biases and has been available for a long period of time, its uptake has been slow or almost nonexistent within the scientific community. In this chapter, we propose a methodology to integrate de novo peptide sequencing using three commonly available software solutions in tandem, complemented by homology searching, and manual validation of spectra. This simplified method would allow greater use of de novo sequencing approaches and potentially greatly increase proteome coverage leading to the unearthing of valuable insights into protein biology, especially of organisms whose genomes have been recently sequenced or are poorly annotated.

  2. DeNovoID: a web-based tool for identifying peptides from sequence and mass tags deduced from de novo peptide sequencing by mass spectroscopy.

    Science.gov (United States)

    Halligan, Brian D; Ruotti, Victor; Twigger, Simon N; Greene, Andrew S

    2005-07-01

    One of the core activities of high-throughput proteomics is the identification of peptides from mass spectra. Some peptides can be identified using spectral matching programs like Sequest or Mascot, but many spectra do not produce high quality database matches. De novo peptide sequencing is an approach to determine partial peptide sequences for some of the unidentified spectra. A drawback of de novo peptide sequencing is that it produces a series of ordered and disordered sequence tags and mass tags rather than a complete, non-degenerate peptide amino acid sequence. This incomplete data is difficult to use in conventional search programs such as BLAST or FASTA. DeNovoID is a program that has been specifically designed to use degenerate amino acid sequence and mass data derived from MS experiments to search a peptide database. Since the algorithm employed depends on the amino acid composition of the peptide and not its sequence, DeNovoID does not have to consider all possible sequences, but rather a smaller number of compositions consistent with a spectrum. DeNovoID also uses a geometric indexing scheme that reduces the number of calculations required to determine the best peptide match in the database. DeNovoID is available at http://proteomics.mcw.edu/denovoid.

  3. A high-throughput de novo sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry

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    Banfield Jillian F

    2010-03-01

    Full Text Available Abstract Background High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate de novo sequencing for identification of post-translational modifications and amino acid polymorphisms. Results In this study, a new de novo sequencing algorithm, called Vonode, has been developed specifically for analysis of such high-resolution tandem mass spectra. To fully exploit the high mass accuracy of these spectra, a unique scoring system is proposed to evaluate sequence tags based primarily on mass accuracy information of fragment ions. Consensus sequence tags were inferred for 11,422 spectra with an average peptide length of 5.5 residues from a total of 40,297 input spectra acquired in a 24-hour proteomics measurement of Rhodopseudomonas palustris. The accuracy of inferred consensus sequence tags was 84%. According to our comparison, the performance of Vonode was shown to be superior to the PepNovo v2.0 algorithm, in terms of the number of de novo sequenced spectra and the sequencing accuracy. Conclusions Here, we improved de novo sequencing performance by developing a new algorithm specifically for high-resolution tandem mass spectral data. The Vonode algorithm is freely available for download at http://compbio.ornl.gov/Vonode.

  4. De novo assembly and characterization of the Trichuris trichiura adult worm transcriptome using Ion Torrent sequencing.

    Science.gov (United States)

    Santos, Leonardo N; Silva, Eduardo S; Santos, André S; De Sá, Pablo H; Ramos, Rommel T; Silva, Artur; Cooper, Philip J; Barreto, Maurício L; Loureiro, Sebastião; Pinheiro, Carina S; Alcantara-Neves, Neuza M; Pacheco, Luis G C

    2016-07-01

    Infection with helminthic parasites, including the soil-transmitted helminth Trichuris trichiura (human whipworm), has been shown to modulate host immune responses and, consequently, to have an impact on the development and manifestation of chronic human inflammatory diseases. De novo derivation of helminth proteomes from sequencing of transcriptomes will provide valuable data to aid identification of parasite proteins that could be evaluated as potential immunotherapeutic molecules in near future. Herein, we characterized the transcriptome of the adult stage of the human whipworm T. trichiura, using next-generation sequencing technology and a de novo assembly strategy. Nearly 17.6 million high-quality clean reads were assembled into 6414 contiguous sequences, with an N50 of 1606bp. In total, 5673 protein-encoding sequences were confidentially identified in the T. trichiura adult worm transcriptome; of these, 1013 sequences represent potential newly discovered proteins for the species, most of which presenting orthologs already annotated in the related species T. suis. A number of transcripts representing probable novel non-coding transcripts for the species T. trichiura were also identified. Among the most abundant transcripts, we found sequences that code for proteins involved in lipid transport, such as vitellogenins, and several chitin-binding proteins. Through a cross-species expression analysis of gene orthologs shared by T. trichiura and the closely related parasites T. suis and T. muris it was possible to find twenty-six protein-encoding genes that are consistently highly expressed in the adult stages of the three helminth species. Additionally, twenty transcripts could be identified that code for proteins previously detected by mass spectrometry analysis of protein fractions of the whipworm somatic extract that present immunomodulatory activities. Five of these transcripts were amongst the most highly expressed protein-encoding sequences in the T

  5. De novo sequencing and analysis of the Ulva linza transcriptome to discover putative mechanisms associated with its successful colonization of coastal ecosystems

    Directory of Open Access Journals (Sweden)

    Zhang Xiaowen

    2012-10-01

    Full Text Available Abstract Background The green algal genus Ulva Linnaeus (Ulvaceae, Ulvales, Chlorophyta is well known for its wide distribution in marine, freshwater, and brackish environments throughout the world. The Ulva species are also highly tolerant of variations in salinity, temperature, and irradiance and are the main cause of green tides, which can have deleterious ecological effects. However, limited genomic information is currently available in this non-model and ecologically important species. Ulva linza is a species that inhabits bedrock in the mid to low intertidal zone, and it is a major contributor to biofouling. Here, we presented the global characterization of the U. linza transcriptome using the Roche GS FLX Titanium platform, with the aim of uncovering the genomic mechanisms underlying rapid and successful colonization of the coastal ecosystems. Results De novo assembly of 382,884 reads generated 13,426 contigs with an average length of 1,000 bases. Contiguous sequences were further assembled into 10,784 isotigs with an average length of 1,515 bases. A total of 304,101 reads were nominally identified by BLAST; 4,368 isotigs were functionally annotated with 13,550 GO terms, and 2,404 isotigs having enzyme commission (EC numbers were assigned to 262 KEGG pathways. When compared with four other full sequenced green algae, 3,457 unique isotigs were found in U. linza and 18 conserved in land plants. In addition, a specific photoprotective mechanism based on both LhcSR and PsbS proteins and a C4-like carbon-concentrating mechanism were found, which may help U. linza survive stress conditions. At least 19 transporters for essential inorganic nutrients (i.e., nitrogen, phosphorus, and sulphur were responsible for its ability to take up inorganic nutrients, and at least 25 eukaryotic cytochrome P450s, which is a higher number than that found in other algae, may be related to their strong allelopathy. Multi-origination of the stress related proteins

  6. De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora).

    Science.gov (United States)

    Liu, Le; Zhang, Shijie; Lian, Chunlan

    2015-12-04

    Japanese red pine (Pinus densiflora) is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98%) unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%), Amborella trichopoda (9.83%), and Pinus taeda (4.15%). A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite) software, of which the tri-nucleotide repeats were most abundant (50.18%) and 629 EST-SSR (expressed sequence tag- simple sequence repeats) primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s) mapping for pine for P. densiflora.

  7. De Novo Transcriptome Sequencing Analysis of cDNA Library and Large-Scale Unigene Assembly in Japanese Red Pine (Pinus densiflora

    Directory of Open Access Journals (Sweden)

    Le Liu

    2015-12-01

    Full Text Available Japanese red pine (Pinus densiflora is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 (64.98% unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis (41.60%, Amborella trichopoda (9.83%, and Pinus taeda (4.15%. A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA (MicroSAtellite software, of which the tri-nucleotide repeats were most abundant (50.18% and 629 EST-SSR (expressed sequence tag- simple sequence repeats primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related gene(s mapping for pine for P. densiflora.

  8. De novo transcriptome sequencing of axolotl blastema for identification of differentially expressed genes during limb regeneration

    Science.gov (United States)

    2013-01-01

    Background Salamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing. Results The sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization. Conclusion The results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration. PMID:23815514

  9. Qualitative de novo analysis of full length cDNA and quantitative analysis of gene expression for common marmoset (Callithrix jacchus) transcriptomes using parallel long-read technology and short-read sequencing.

    Science.gov (United States)

    Shimizu, Makiko; Iwano, Shunsuke; Uno, Yasuhiro; Uehara, Shotaro; Inoue, Takashi; Murayama, Norie; Onodera, Jun; Sasaki, Erika; Yamazaki, Hiroshi

    2014-01-01

    The common marmoset (Callithrix jacchus) is a non-human primate that could prove useful as human pharmacokinetic and biomedical research models. The cytochromes P450 (P450s) are a superfamily of enzymes that have critical roles in drug metabolism and disposition via monooxygenation of a broad range of xenobiotics; however, information on some marmoset P450s is currently limited. Therefore, identification and quantitative analysis of tissue-specific mRNA transcripts, including those of P450s and flavin-containing monooxygenases (FMO, another monooxygenase family), need to be carried out in detail before the marmoset can be used as an animal model in drug development. De novo assembly and expression analysis of marmoset transcripts were conducted with pooled liver, intestine, kidney, and brain samples from three male and three female marmosets. After unique sequences were automatically aligned by assembling software, the mean contig length was 718 bp (with a standard deviation of 457 bp) among a total of 47,883 transcripts. Approximately 30% of the total transcripts were matched to known marmoset sequences. Gene expression in 18 marmoset P450- and 4 FMO-like genes displayed some tissue-specific patterns. Of these, the three most highly expressed in marmoset liver were P450 2D-, 2E-, and 3A-like genes. In extrahepatic tissues, including brain, gene expressions of these monooxygenases were lower than those in liver, although P450 3A4 (previously P450 3A21) in intestine and P450 4A11- and FMO1-like genes in kidney were relatively highly expressed. By means of massive parallel long-read sequencing and short-read technology applied to marmoset liver, intestine, kidney, and brain, the combined next-generation sequencing analyses reported here were able to identify novel marmoset drug-metabolizing P450 transcripts that have until now been little reported. These results provide a foundation for mechanistic studies and pave the way for the use of marmosets as model animals

  10. DIME: a novel framework for de novo metagenomic sequence assembly.

    Science.gov (United States)

    Guo, Xuan; Yu, Ning; Ding, Xiaojun; Wang, Jianxin; Pan, Yi

    2015-02-01

    The recently developed next generation sequencing platforms not only decrease the cost for metagenomics data analysis, but also greatly enlarge the size of metagenomic sequence datasets. A common bottleneck of available assemblers is that the trade-off between the noise of the resulting contigs and the gain in sequence length for better annotation has not been attended enough for large-scale sequencing projects, especially for the datasets with low coverage and a large number of nonoverlapping contigs. To address this limitation and promote both accuracy and efficiency, we develop a novel metagenomic sequence assembly framework, DIME, by taking the DIvide, conquer, and MErge strategies. In addition, we give two MapReduce implementations of DIME, DIME-cap3 and DIME-genovo, on Apache Hadoop platform. For a systematic comparison of the performance of the assembly tasks, we tested DIME and five other popular short read assembly programs, Cap3, Genovo, MetaVelvet, SOAPdenovo, and SPAdes on four synthetic and three real metagenomic sequence datasets with various reads from fifty thousand to a couple million in size. The experimental results demonstrate that our method not only partitions the sequence reads with an extremely high accuracy, but also reconstructs more bases, generates higher quality assembled consensus, and yields higher assembly scores, including corrected N50 and BLAST-score-per-base, than other tools with a nearly theoretical speed-up. Results indicate that DIME offers great improvement in assembly across a range of sequence abundances and thus is robust to decreasing coverage.

  11. A framework for the detection of de novo mutations in family-based sequencing data

    Science.gov (United States)

    Francioli, Laurent C; Cretu-Stancu, Mircea; Garimella, Kiran V; Fromer, Menachem; Kloosterman, Wigard P; Wijmenga, Cisca; Investigator, Principal; Swertz, Morris A; van Duijn, Cornelia M; Boomsma, Dorret I; Slagboom, PEline; van Ommen, Gertjan B; de Bakker, Paul IW; Swertz, Morris A; Francioli, Laurent C; van Dijk, Freerk; Menelaou, Androniki; Neerincx, Pieter BT; Pulit, Sara L; Deelen, Patrick; Elbers, Clara C; Francesco Palamara, Pier; Pe'er, Itsik; Abdellaoui, Abdel; Kloosterman, Wigard P; van Oven, Mannis; Vermaat, Martijn; Li, Mingkun; Laros, Jeroen FJ; Stoneking, Mark; de Knijff, Peter; Kayser, Manfred; Veldink, Jan H; van den Berg, Leonard H; Byelas, Heorhiy; den Dunnen, Johan T; Dijkstra, Martijn; Amin, Najaf; van der Velde, K Joeri; Hottenga, Jouke Jan; van Setten, Jessica; van Leeuwen, Elisabeth M; Kanterakis, Alexandros; Kattenberg, Mathijs; Karssen, Lennart C; van Schaik, Barbera DC; Bot, Jan; Nijman, Isaäc J; Renkens, Ivo; van Enckevort, David; Mei, Hailiang; Koval, Vyacheslav; Estrada, Karol; Medina-Gomez, Carolina; Ye, Kai; Lameijer, Eric-Wubbo; Moed, Matthijs H; Hehir-Kwa, Jayne Y; Handsaker, Robert E; McCarroll, Steven A; Sunyaev, Shamil R; Polak, Paz; Vuzman, Dana; Sohail, Mashaal; Hormozdiari, Fereydoun; Marschall, Tobias; Schönhuth, Alexander; Guryev, Victor; de Bakker, Paul IW; Slagboom, P Eline; Beekman, Marian B; de Craen, Anton JM; Suchiman, H Eka D; Hofman, Albert; van Duijn, Cornelia M; Oostra, Ben; Isaacs, Aaron; Amin, Najaf; Rivadeneira, Fernando; Uitterlinden, André G; Boomsma, Dorret I; Willemsen, Gonneke; Platteel, Mathieu; Pitts, Steven J; Potluri, Shobha; Sundar, Purnima; Cox, David R; Li, Qibin; Li, Yingrui; Du, Yuanping; Chen, Ruoyan; Cao, Hongzhi; Li, Ning; Cao, Sujie; Wang, Jun; Bovenberg, Jasper A; Brandsma, Margreet; Samocha, Kaitlin E; Neale, Benjamin M; Daly, Mark J; Banks, Eric; DePristo, Mark A; de Bakker, Paul IW

    2017-01-01

    Germline mutation detection from human DNA sequence data is challenging due to the rarity of such events relative to the intrinsic error rates of sequencing technologies and the uneven coverage across the genome. We developed PhaseByTransmission (PBT) to identify de novo single nucleotide variants and short insertions and deletions (indels) from sequence data collected in parent-offspring trios. We compute the joint probability of the data given the genotype likelihoods in the individual family members, the known familial relationships and a prior probability for the mutation rate. Candidate de novo mutations (DNMs) are reported along with their posterior probability, providing a systematic way to prioritize them for validation. Our tool is integrated in the Genome Analysis Toolkit and can be used together with the ReadBackedPhasing module to infer the parental origin of DNMs based on phase-informative reads. Using simulated data, we show that PBT outperforms existing tools, especially in low coverage data and on the X chromosome. We further show that PBT displays high validation rates on empirical parent-offspring sequencing data for whole-exome data from 104 trios and X-chromosome data from 249 parent-offspring families. Finally, we demonstrate an association between father's age at conception and the number of DNMs in female offspring's X chromosome, consistent with previous literature reports. PMID:27876817

  12. Whole-Genome de novo Sequencing Of Quail And Grey Partridge

    DEFF Research Database (Denmark)

    Holm, Lars-Erik; Panitz, Frank; Burt, Dave;

    2011-01-01

    The development in sequencing methods has made it possible to perform whole genome de novo sequencing of species without large commercial interests. Within the EU-financed QUANTOMICS project (KBBE-2A-222664), we have performed de novo sequencing of quail (Coturnix coturnix) and grey partridge...... comparative studies towards the chicken genome and will aid in identifying evolutionarily conserved sequences within the Galliformes. The obtained sequences from quail and partridge represent a beginning of generating the whole genome sequence for these species. The continuation of establishing the genome...

  13. Anchored pseudo-de novo assembly of human genomes identifies extensive sequence variation from unmapped sequence reads.

    Science.gov (United States)

    Faber-Hammond, Joshua J; Brown, Kim H

    2016-07-01

    The human genome reference (HGR) completion marked the genomics era beginning, yet despite its utility universal application is limited by the small number of individuals used in its development. This is highlighted by the presence of high-quality sequence reads failing to map within the HGR. Sequences failing to map generally represent 2-5 % of total reads, which may harbor regions that would enhance our understanding of population variation, evolution, and disease. Alternatively, complete de novo assemblies can be created, but these effectively ignore the groundwork of the HGR. In an effort to find a middle ground, we developed a bioinformatic pipeline that maps paired-end reads to the HGR as separate single reads, exports unmappable reads, de novo assembles these reads per individual and then combines assemblies into a secondary reference assembly used for comparative analysis. Using 45 diverse 1000 Genomes Project individuals, we identified 351,361 contigs covering 195.5 Mb of sequence unincorporated in GRCh38. 30,879 contigs are represented in multiple individuals with ~40 % showing high sequence complexity. Genomic coordinates were generated for 99.9 %, with 52.5 % exhibiting high-quality mapping scores. Comparative genomic analyses with archaic humans and primates revealed significant sequence alignments and comparisons with model organism RefSeq gene datasets identified novel human genes. If incorporated, these sequences will expand the HGR, but more importantly our data highlight that with this method low coverage (~10-20×) next-generation sequencing can still be used to identify novel unmapped sequences to explore biological functions contributing to human phenotypic variation, disease and functionality for personal genomic medicine.

  14. De novo sequencing and comparative analysis of leaf transcriptomes of diverse condensed tannin-containing lines of underutilized Psophocarpus tetragonolobus (L.) DC

    Science.gov (United States)

    Singh, Vinayak; Goel, Ridhi; Pande, Veena; Asif, Mehar Hasan; Mohanty, Chandra Sekhar

    2017-01-01

    Condensed tannin (CT) or proanthocyanidin (PA) is a unique group of phenolic metabolite with high molecular weight with specific structure. It is reported that, the presence of high-CT in the legumes adversely affect the nutrients in the plant and impairs the digestibility upon consumption by animals. Winged bean (Psophocarpus tetragonolobus (L.) DC.) is one of the promising underutilized legume with high protein and oil-content. One of the reasons for its underutilization is due to the presence of CT. Transcriptome sequencing of leaves of two diverse CT-containing lines of P. tetragonolobus was carried out on Illumina Nextseq 500 sequencer to identify the underlying genes and contigs responsible for CT-biosynthesis. RNA-Seq data generated 102586 and 88433 contigs for high (HCTW) and low CT (LCTW) lines of P. tetragonolobus, respectively. Based on the similarity searches against gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) database revealed 5210 contigs involved in 229 different pathways. A total of 1235 contigs were detected to differentially express between HCTW and LCTW lines. This study along with its findings will be helpful in providing information for functional and comparative genomic analysis of condensed tannin biosynthesis in this plant in specific and legumes in general. PMID:28322296

  15. Whole Exome Sequencing for a Patient with Rubinstein-Taybi Syndrome Reveals de Novo Variants besides an Overt CREBBP Mutation

    Directory of Open Access Journals (Sweden)

    Hee Jeong Yoo

    2015-03-01

    Full Text Available Rubinstein-Taybi syndrome (RSTS is a rare condition with a prevalence of 1 in 125,000–720,000 births and characterized by clinical features that include facial, dental, and limb dysmorphology and growth retardation. Most cases of RSTS occur sporadically and are caused by de novo mutations. Cytogenetic or molecular abnormalities are detected in only 55% of RSTS cases. Previous genetic studies have yielded inconsistent results due to the variety of methods used for genetic analysis. The purpose of this study was to use whole exome sequencing (WES to evaluate the genetic causes of RSTS in a young girl presenting with an Autism phenotype. We used the Autism diagnostic observation schedule (ADOS and Autism diagnostic interview revised (ADI-R to confirm her diagnosis of Autism. In addition, various questionnaires were used to evaluate other psychiatric features. We used WES to analyze the DNA sequences of the patient and her parents and to search for de novo variants. The patient showed all the typical features of Autism, WES revealed a de novo frameshift mutation in CREBBP and de novo sequence variants in TNC and IGFALS genes. Mutations in the CREBBP gene have been extensively reported in RSTS patients, while potential missense mutations in TNC and IGFALS genes have not previously been associated with RSTS. The TNC and IGFALS genes are involved in central nervous system development and growth. It is possible for patients with RSTS to have additional de novo variants that could account for previously unexplained phenotypes.

  16. Stacks: building and genotyping Loci de novo from short-read sequences.

    Science.gov (United States)

    Catchen, Julian M; Amores, Angel; Hohenlohe, Paul; Cresko, William; Postlethwait, John H

    2011-08-01

    Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress. Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks can recover thousands of single nucleotide polymorphism (SNP) markers useful for the genetic analysis of crosses or populations. Stacks can generate markers for ultra-dense genetic linkage maps, facilitate the examination of population phylogeography, and help in reference genome assembly. We report here the algorithms implemented in Stacks and demonstrate their efficacy by constructing loci from simulated RAD-tags taken from the stickleback reference genome and by recapitulating and improving a genetic map of the zebrafish, Danio rerio.

  17. Evaluating de novo sequencing in proteomics: already an accurate alternative to database-driven peptide identification?

    Science.gov (United States)

    Muth, Thilo; Renard, Bernhard Y

    2017-03-21

    While peptide identifications in mass spectrometry (MS)-based shotgun proteomics are mostly obtained using database search methods, high-resolution spectrum data from modern MS instruments nowadays offer the prospect of improving the performance of computational de novo peptide sequencing. The major benefit of de novo sequencing is that it does not require a reference database to deduce full-length or partial tag-based peptide sequences directly from experimental tandem mass spectrometry spectra. Although various algorithms have been developed for automated de novo sequencing, the prediction accuracy of proposed solutions has been rarely evaluated in independent benchmarking studies. The main objective of this work is to provide a detailed evaluation on the performance of de novo sequencing algorithms on high-resolution data. For this purpose, we processed four experimental data sets acquired from different instrument types from collision-induced dissociation and higher energy collisional dissociation (HCD) fragmentation mode using the software packages Novor, PEAKS and PepNovo. Moreover, the accuracy of these algorithms is also tested on ground truth data based on simulated spectra generated from peak intensity prediction software. We found that Novor shows the overall best performance compared with PEAKS and PepNovo with respect to the accuracy of correct full peptide, tag-based and single-residue predictions. In addition, the same tool outpaced the commercial competitor PEAKS in terms of running time speedup by factors of around 12-17. Despite around 35% prediction accuracy for complete peptide sequences on HCD data sets, taken as a whole, the evaluated algorithms perform moderately on experimental data but show a significantly better performance on simulated data (up to 84% accuracy). Further, we describe the most frequently occurring de novo sequencing errors and evaluate the influence of missing fragment ion peaks and spectral noise on the accuracy. Finally

  18. De Novo transcriptome analysis of Oncomelania hupensis after molluscicide treatment by next-generation sequencing: implications for biology and future snail interventions.

    Directory of Open Access Journals (Sweden)

    Qin Ping Zhao

    Full Text Available The freshwater snail Oncomelania hupensis is the only intermediate host of Schistosoma japonicum, which causes schistosomiasis. This disease is endemic in the Far East, especially in mainland China. Because niclosamide is the only molluscicide recommended by the World Health Organization, 50% wettable powder of niclosamide ethanolamine salt (WPN, the only chemical molluscicide available in China, has been widely used as the main snail control method for over two decades. Recently, a novel molluscicide derived from niclosamide, the salt of quinoid-2',5-dichloro-4'-nitro-salicylanilide (Liu Dai Shui Yang An, LDS, has been developed and proven to have the same molluscicidal effect as WPN, with lower cost and significantly lower toxicity to fish than WPN. The mechanism by which these molluscicides cause snail death is not known. Here, we report the next-generation transcriptome sequencing of O. hupensis; 145,008,667 clean reads were generated and assembled into 254,286 unigenes. Using GO and KEGG databases, 14,860 unigenes were assigned GO annotations and 4,686 unigenes were mapped to 250 KEGG pathways. Many sequences involved in key processes associated with biological regulation and innate immunity have been identified. After the snails were exposed to LDS and WPN, 254 unigenes showed significant differential expression. These genes were shown to be involved in cell structure defects and the inhibition of neurohumoral transmission and energy metabolism, which may cause snail death. Gene expression patterns differed after exposure to LDS and WPN, and these differences must be elucidated by the identification and annotation of these unknown unigenes. We believe that this first large-scale transcriptome dataset for O. hupensis will provide an opportunity for the in-depth analysis of this biomedically important freshwater snail at the molecular level and accelerate studies of the O. hupensis genome. The data elucidating the molluscicidal mechanism

  19. [De novo sequencing and analysis of root transcriptome to reveal regulation of gene expression by moderate drought stress in Glycyrrhiza uralensis].

    Science.gov (United States)

    Zhang, Chun-rong; Sang, Xue-yu; Qu, Meng; Tang, Xiao-min; Cheng, Xuan-xuan; Pan, Li-ming; Yang, Quan

    2015-12-01

    Moderate drought stress has been found to promote the accumulation of active ingredients in Glycyrrhiza uralensis root and hence improve the medicinal quality. In this study, the transcriptomes of 6-month-old moderate drought stressed and control G. uralensis root (the relative water content in soil was 40%-45% and 70%-75%, respectively) were sequenced using Illumina HiSeq 2000. A total of 80,490 490 and 82 588 278 clean reads, 94,828 and 305,100 unigenes with N50 sequence of 1,007 and 1,125 nt were obtained in drought treated and control transcriptome, respectively. Differentially expressed genes analysis revealed that the genes of some cell wall enzymes such as β-xylosidase, legumain and GDP-L-fucose synthase were down-regulated indicating that moderate drought stress might inhibit the primary cell wall degradation and programmed cell death in root cells. The genes of some key enzymes involved in terpenoid and flavonoid biosynthesis were up-regulated by moderate drought stress might be the reason for the enhancement for the active ingredients accumulation in G. uralensis root. The promotion of the biosynthesis and signal transduction of auxin, ethylene and cytokinins by moderate drought stress might enhance the root formation and cell proliferation. The promotion of the biosynthesis and signal transduction of abscisic acid and jasmonic acid by moderate drought stress might enhance the drought stress tolerance in G. uralensis. The inhibition of the biosynthesis and signal transduction of gibberellin and brassinolide by moderate drought stress might retard the shoot growth in G. uralensis.

  20. Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

    Science.gov (United States)

    Christen, Matthias; Deutsch, Samuel; Christen, Beat

    2015-08-21

    Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

  1. De Novo Sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, Reveal a Variable Genomic Landscape

    Directory of Open Access Journals (Sweden)

    Benjamin J. Tully

    2015-01-01

    Full Text Available Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.

  2. A hybrid approach for de novo human genome sequence assembly and phasing.

    Science.gov (United States)

    Mostovoy, Yulia; Levy-Sakin, Michal; Lam, Jessica; Lam, Ernest T; Hastie, Alex R; Marks, Patrick; Lee, Joyce; Chu, Catherine; Lin, Chin; Džakula, Željko; Cao, Han; Schlebusch, Stephen A; Giorda, Kristina; Schnall-Levin, Michael; Wall, Jeffrey D; Kwok, Pui-Yan

    2016-07-01

    Despite tremendous progress in genome sequencing, the basic goal of producing a phased (haplotype-resolved) genome sequence with end-to-end contiguity for each chromosome at reasonable cost and effort is still unrealized. In this study, we describe an approach to performing de novo genome assembly and experimental phasing by integrating the data from Illumina short-read sequencing, 10X Genomics linked-read sequencing, and BioNano Genomics genome mapping to yield a high-quality, phased, de novo assembled human genome.

  3. De novo sequencing and comparative transcriptome analysis of white petals and red labella in Phalaenopsis for discovery of genes related to flower color and floral differentation

    Directory of Open Access Journals (Sweden)

    Yuxia Yang

    2014-09-01

    Full Text Available Phalaenopsis is one of the world’s most popular and important epiphytic monopodial orchids. The extraordinary floral diversity of Phalaenopsis is a reflection of its evolutionary success. As a consequence of this diversity, and of the complexity of flower color development in Phalaenopsis, this species is a valuable research material for developmental biology studies. Nevertheless, research on the molecular mechanisms underlying flower color and floral organ formation in Phalaenopsis is still in the early phases. In this study, we generated large amounts of data from Phalaenopsis flowers by combining Illumina sequencing with differentially expressed gene (DEG analysis. We obtained 37 723 and 34 020 unigenes from petals and labella, respectively. A total of 2736 DEGs were identified, and the functions of many DEGs were annotated by BLAST-searching against several public databases. We mapped 837 up-regulated DEGs (432 from petals and 405 from labella to 102 Kyoto Encyclopedia of Genes and Genomes pathways. Almost all pathways were represented in both petals (102 pathways and labella (99 pathways. DEGs involved in energy metabolism were significantly differentially distributed between labella and petals, and various DEGs related to flower color and floral differentiation were found in the two organs. Interestingly, we also identified genes encoding several key enzymes involved in carotenoid synthesis. These genes were differentially expressed between petals and labella, suggesting that carotenoids may influence Phalaenopsis flower color. We thus conclude that a combination of anthocyanins and/or carotenoids determine flower color formation in Phalaenopsis. These results broaden our understanding of the mechanisms controlling flower color and floral organ differentiation in Phalaenopsis and other orchids.

  4. De novo transcriptome sequencing of Acer palmatum and comprehensive analysis of differentially expressed genes under salt stress in two contrasting genotypes.

    Science.gov (United States)

    Rong, Liping; Li, Qianzhong; Li, Shushun; Tang, Ling; Wen, Jing

    2016-04-01

    Maple (Acer palmatum) is an important species for landscape planting worldwide. Salt stress affects the normal growth of the Maple leaf directly, leading to loss of esthetic value. However, the limited availability of Maple genomic information has hindered research on the mechanisms underlying this tolerance. In this study, we performed comprehensive analyses of the salt tolerance in two genotypes of Maple using RNA-seq. Approximately 146.4 million paired-end reads, representing 181,769 unigenes, were obtained. The N50 length of the unigenes was 738 bp, and their total length over 102.66 Mb. 14,090 simple sequence repeats and over 500,000 single nucleotide polymorphisms were identified, which represent useful resources for marker development. Importantly, 181,769 genes were detected in at least one library, and 303 differentially expressed genes (DEGs) were identified between salt-sensitive and salt-tolerant genotypes. Among these DEGs, 125 were upregulated and 178 were downregulated genes. Two MYB-related proteins and one LEA protein were detected among the first 10 most downregulated genes. Moreover, a methyltransferase-related gene was detected among the first 10 most upregulated genes. The three most significantly enriched pathways were plant hormone signal transduction, arginine and proline metabolism, and photosynthesis. The transcriptome analysis provided a rich genetic resource for gene discovery related to salt tolerance in Maple, and in closely related species. The data will serve as an important public information platform to further our understanding of the molecular mechanisms involved in salt tolerance in Maple.

  5. Effects of GC bias in next-generation-sequencing data on de novo genome assembly.

    Science.gov (United States)

    Chen, Yen-Chun; Liu, Tsunglin; Yu, Chun-Hui; Chiang, Tzen-Yuh; Hwang, Chi-Chuan

    2013-01-01

    Next-generation-sequencing (NGS) has revolutionized the field of genome assembly because of its much higher data throughput and much lower cost compared with traditional Sanger sequencing. However, NGS poses new computational challenges to de novo genome assembly. Among the challenges, GC bias in NGS data is known to aggravate genome assembly. However, it is not clear to what extent GC bias affects genome assembly in general. In this work, we conduct a systematic analysis on the effects of GC bias on genome assembly. Our analyses reveal that GC bias only lowers assembly completeness when the degree of GC bias is above a threshold. At a strong GC bias, the assembly fragmentation due to GC bias can be explained by the low coverage of reads in the GC-poor or GC-rich regions of a genome. This effect is observed for all the assemblers under study. Increasing the total amount of NGS data thus rescues the assembly fragmentation because of GC bias. However, the amount of data needed for a full rescue depends on the distribution of GC contents. Both low and high coverage depths due to GC bias lower the accuracy of assembly. These pieces of information provide guidance toward a better de novo genome assembly in the presence of GC bias.

  6. De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology.

    Science.gov (United States)

    Nijkamp, Jurgen F; van den Broek, Marcel; Datema, Erwin; de Kok, Stefan; Bosman, Lizanne; Luttik, Marijke A; Daran-Lapujade, Pascale; Vongsangnak, Wanwipa; Nielsen, Jens; Heijne, Wilbert H M; Klaassen, Paul; Paddon, Chris J; Platt, Darren; Kötter, Peter; van Ham, Roeland C; Reinders, Marcel J T; Pronk, Jack T; de Ridder, Dick; Daran, Jean-Marc

    2012-03-26

    Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.

  7. De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology

    NARCIS (Netherlands)

    Nijkamp, J.F.; Van den Broek, M.A.; Datema, E.; De Kok, S.; Bosman, L.; Luttik, M.A.H.; Daran-Lapujade, P.A.S.; Vongsangnak, W.; Nielsen, J.; Heijne. W.H.M.; Klaassen, P.; Paddon, C.J.; Platt, D.; Kötter, P.; Van Ham, R.C.; Reinders, M.J.T.; Pronk, J.T.; De Ridder, D.; Daran, J.M.

    2012-01-01

    Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization

  8. De novo sequencing, assembly, and analysis of the root transcriptome of Persea americana (Mill.) in response to Phytophthora cinnamomi and flooding.

    Science.gov (United States)

    Reeksting, Bianca J; Coetzer, Nanette; Mahomed, Waheed; Engelbrecht, Juanita; van den Berg, Noëlani

    2014-01-01

    Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR) causes damage to the feeder roots of trees, causing necrosis. This leads to branch-dieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies.

  9. De novo sequencing, assembly, and analysis of the root transcriptome of Persea americana (Mill. in response to Phytophthora cinnamomi and flooding.

    Directory of Open Access Journals (Sweden)

    Bianca J Reeksting

    Full Text Available Avocado is a diploid angiosperm containing 24 chromosomes with a genome estimated to be around 920 Mb. It is an important fruit crop worldwide but is susceptible to a root rot caused by the ubiquitous oomycete Phytophthora cinnamomi. Phytophthora root rot (PRR causes damage to the feeder roots of trees, causing necrosis. This leads to branch-dieback and eventual tree death, resulting in severe losses in production. Control strategies are limited and at present an integrated approach involving the use of phosphite, tolerant rootstocks, and proper nursery management has shown the best results. Disease progression of PRR is accelerated under high soil moisture or flooding conditions. In addition, avocado is highly susceptible to flooding, with even short periods of flooding causing significant losses. Despite the commercial importance of avocado, limited genomic resources are available. Next generation sequencing has provided the means to generate sequence data at a relatively low cost, making this an attractive option for non-model organisms such as avocado. The aims of this study were to generate sequence data for the avocado root transcriptome and identify stress-related genes. Tissue was isolated from avocado infected with P. cinnamomi, avocado exposed to flooding and avocado exposed to a combination of these two stresses. Three separate sequencing runs were performed on the Roche 454 platform and produced approximately 124 Mb of data. This was assembled into 7685 contigs, with 106 448 sequences remaining as singletons. Genes involved in defence pathways such as the salicylic acid and jasmonic acid pathways as well as genes associated with the response to low oxygen caused by flooding, were identified. This is the most comprehensive study of transcripts derived from root tissue of avocado to date and will provide a useful resource for future studies.

  10. De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology

    Directory of Open Access Journals (Sweden)

    Nijkamp Jurgen F

    2012-03-01

    Full Text Available Abstract Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV, insertions/deletions (indels and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.

  11. Genomic Resources for Water Yam (Dioscorea alata L.): Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries.

    Science.gov (United States)

    Saski, Christopher A; Bhattacharjee, Ranjana; Scheffler, Brian E; Asiedu, Robert

    2015-01-01

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp.) is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST)-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS) profiles on two yam (Dioscorea alata L.) genotypes (TDa 95/00328 and TDa 95-310) was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using different approaches

  12. Genomic Resources for Water Yam (Dioscorea alata L.: Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries.

    Directory of Open Access Journals (Sweden)

    Christopher A Saski

    Full Text Available The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp. is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS profiles on two yam (Dioscorea alata L. genotypes (TDa 95/00328 and TDa 95-310 was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using

  13. Whole-genome sequencing for comparative genomics and de novo genome assembly.

    Science.gov (United States)

    Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

    2015-01-01

    Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

  14. De Novo Sequencing and Analysis of the Safflower Transcriptome to Discover Putative Genes Associated with Safflor Yellow in Carthamus tinctorius L.

    Directory of Open Access Journals (Sweden)

    Xiuming Liu

    2015-10-01

    Full Text Available Safflower (Carthamus tinctorius L., an important traditional Chinese medicine, is cultured widely for its pharmacological effects, but little is known regarding the genes related to the metabolic regulation of the safflower’s yellow pigment. To investigate genes related to safflor yellow biosynthesis, 454 pyrosequencing of flower RNA at different developmental stages was performed, generating large databases.In this study, we analyzed 454 sequencing data from different flowering stages in safflower. In total, 1,151,324 raw reads and 1,140,594 clean reads were produced, which were assembled into 51,591 unigenes with an average length of 679 bp and a maximum length of 5109 bp. Among the unigenes, 40,139 were in the early group, 39,768 were obtained from the full group and 28,316 were detected in both samples. With the threshold of “log2 ratio ≥ 1”, there were 34,464 differentially expressed genes, of which 18,043 were up-regulated and 16,421 were down-regulated in the early flower library. Based on the annotations of the unigenes, 281 pathways were predicted. We selected 12 putative genes and analyzed their expression levels using quantitative real time-PCR. The results were consistent with the 454 sequencing results. In addition, the expression of chalcone synthase, chalcone isomerase and anthocyanidin synthase, which are involved in safflor yellow biosynthesis and safflower yellow pigment (SYP content, were analyzed in different flowering periods, indicating that their expression levels were related to SYP synthesis. Moreover, to further confirm the results of the 454 pyrosequencing, full-length cDNA of chalcone isomerase (CHI and anthocyanidin synthase (ANS were cloned from safflower petal by RACE (Rapid-amplification of cDNA ends method according to fragment of the transcriptome.

  15. De novo sequence analysis of cytochrome P450 1-3 genes expressed in ostrich liver with highest expression of CYP2G19.

    Science.gov (United States)

    Kawai, Yusuke K; Watanabe, Kensuke P; Ishii, Akihiro; Ohnuma, Aiko; Sawa, Hirofumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2013-09-01

    The cytochrome P450 (CYP) 1-3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1-3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2-10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.

  16. De novo assembly of human genomes with massively parallel short read sequencing

    DEFF Research Database (Denmark)

    Li, Ruiqiang; Zhu, Hongmei; Ruan, Jue

    2010-01-01

    genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities...... for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way....

  17. REPdenovo: Inferring De Novo Repeat Motifs from Short Sequence Reads.

    Directory of Open Access Journals (Sweden)

    Chong Chu

    Full Text Available Repeat elements are important components of eukaryotic genomes. One limitation in our understanding of repeat elements is that most analyses rely on reference genomes that are incomplete and often contain missing data in highly repetitive regions that are difficult to assemble. To overcome this problem we develop a new method, REPdenovo, which assembles repeat sequences directly from raw shotgun sequencing data. REPdenovo can construct various types of repeats that are highly repetitive and have low sequence divergence within copies. We show that REPdenovo is substantially better than existing methods both in terms of the number and the completeness of the repeat sequences that it recovers. The key advantage of REPdenovo is that it can reconstruct long repeats from sequence reads. We apply the method to human data and discover a number of potentially new repeats sequences that have been missed by previous repeat annotations. Many of these sequences are incorporated into various parasite genomes, possibly because the filtering process for host DNA involved in the sequencing of the parasite genomes failed to exclude the host derived repeat sequences. REPdenovo is a new powerful computational tool for annotating genomes and for addressing questions regarding the evolution of repeat families. The software tool, REPdenovo, is available for download at https://github.com/Reedwarbler/REPdenovo.

  18. Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

    Science.gov (United States)

    Saha, Bodhisattwa; Sircar, Gaurab; Pandey, Naren; Gupta Bhattacharya, Swati

    2015-11-06

    Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

  19. De novo assembly and analysis of crow lungs transcriptome.

    Science.gov (United States)

    Vijayakumar, Periyasamy; Raut, Ashwin Ashok; Kumar, Pushpendra; Sharma, Deepak; Mishra, Anamika

    2014-09-01

    The jungle crow (Corvus macrorhynchos) belongs to the order Passeriformes of bird species and is important for avian ecological and evolutionary genetics studies. However, there is limited information on the transcriptome data of this species. In the present study, we report the characterization of the lung transcriptome of the jungle crow using GS FLX Titanium XLR70. Altogether, 1,510,303 high-quality sequence reads with 581,198,230 bases was de novo assembled into 22,169 isotigs (isotig represents an individual transcript) and 784,009 singletons. Using these isotigs and 581,681 length-filtered (greater than 300 bp) singletons, 20,010 unique protein-coding genes were identified by BLASTx comparison against a nonredundant (nr) protein sequence database. Comparative analysis revealed that 46,604 (70.29%) and 51,642 (72.48%) of the assembled transcripts have significant similarity to zebra finch and chicken RefSeq proteins, respectively. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Transcripts putatively involved in the immune response were identified. Furthermore, 20,599 single nucleotide polymorphisms (SNPs) and 7525 simple sequence repeats (SSRs) were retrieved from the assembled transcript database. This resource should lay an important base for future ecological, evolutionary, and conservation genetic studies on this species and in other related species.

  20. De Novo Sequencing of Peptides from Top-Down Tandem Mass Spectra

    Energy Technology Data Exchange (ETDEWEB)

    Vyatkina, Kira; Wu, Si; Dekker, Lennard J. M.; VanDuijn, Martijn M.; Liu, Xiaowen; Tolić, Nikola; Dvorkin, Mikhail; Alexandrova, Sonya; Luider, Theo M.; Paša-Tolić, Ljiljana; Pevzner, Pavel A.

    2015-11-06

    De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technology, now gaining more and more popularity, opens new perspectives for protein analysis and characterization, implying a need in efficient algorithms for processing this kind of MS/MS data. Here we describe a method that allows to retrieve from a set of top-down MS/MS spectra long and accurate sequence fragments of the proteins contained in a sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down datasets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.

  1. De Novo Sequencing of Complex Mixtures of Heparan Sulfate Oligosaccharides

    NARCIS (Netherlands)

    Huang, Rongrong; Zong, Chengli; Venot, Andre; Chiu, Yulun; Zhou, Dandan; Boons, Geert-Jan; Sharp, Joshua S

    2016-01-01

    Here, we describe the first sequencing method of a complex mixture of heparan sulfate tetrasaccharides by LC-MS/MS. Heparin and heparan sulfate (HS) are linear polysaccharides that are modified in a complex manner by N- and O-sulfation, N-acetylation, and epimerization of the uronic acid. Heparin an

  2. SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome.

    Science.gov (United States)

    Stadermann, Kai Bernd; Weisshaar, Bernd; Holtgräwe, Daniela

    2015-09-16

    Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base. We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly. SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly

  3. Sequencing and de novo assembly of the transcriptome of the glassy-winged sharpshooter (Homalodisca vitripennis.

    Directory of Open Access Journals (Sweden)

    Raja Sekhar Nandety

    Full Text Available BACKGROUND: The glassy-winged sharpshooter Homalodisca vitripennis (Hemiptera: Cicadellidae, is a xylem-feeding leafhopper and important vector of the bacterium Xylella fastidiosa; the causal agent of Pierce's disease of grapevines. The functional complexity of the transcriptome of H. vitripennis has not been elucidated thus far. It is a necessary blueprint for an understanding of the development of H. vitripennis and for designing efficient biorational control strategies including those based on RNA interference. RESULTS: Here we elucidate and explore the transcriptome of adult H. vitripennis using high-throughput paired end deep sequencing and de novo assembly. A total of 32,803,656 paired-end reads were obtained with an average transcript length of 624 nucleotides. We assembled 32.9 Mb of the transcriptome of H. vitripennis that spanned across 47,265 loci and 52,708 transcripts. Comparison of our non-redundant database showed that 45% of the deduced proteins of H. vitripennis exhibit identity (e-value ≤1(-5 with known proteins. We assigned Gene Ontology (GO terms, Kyoto Encyclopedia of Genes and Genomes (KEGG annotations, and potential Pfam domains to each transcript isoform. In order to gain insight into the molecular basis of key regulatory genes of H. vitripennis, we characterized predicted proteins involved in the metabolism of juvenile hormone, and biogenesis of small RNAs (Dicer and Piwi sequences from the transcriptomic sequences. Analysis of transposable element sequences of H. vitripennis indicated that the genome is less expanded in comparison to many other insects with approximately 1% of the transcriptome carrying transposable elements. CONCLUSIONS: Our data significantly enhance the molecular resources available for future study and control of this economically important hemipteran. This transcriptional information not only provides a more nuanced understanding of the underlying biological and physiological mechanisms that

  4. In vitro, long-range sequence information for de novo genome assembly via transposase contiguity.

    Science.gov (United States)

    Adey, Andrew; Kitzman, Jacob O; Burton, Joshua N; Daza, Riza; Kumar, Akash; Christiansen, Lena; Ronaghi, Mostafa; Amini, Sasan; Gunderson, Kevin L; Steemers, Frank J; Shendure, Jay

    2014-12-01

    We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to > 1 megabase. These pools are "subhaploid," in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome. The scaffolding approach described here, termed fragScaff, leverages coincidences between the content of different pools as a source of contiguity information. Specifically, CPT-seq data is mapped to a de novo genome assembly, followed by the identification of pairs of contigs or scaffolds whose ends disproportionately co-occur in the same indexed pools, consistent with true adjacency in the genome. Such candidate "joins" are used to construct a graph, which is then resolved by a minimum spanning tree. As a proof-of-concept, we apply CPT-seq and fragScaff to substantially boost the contiguity of de novo assemblies of the human, mouse, and fly genomes, increasing the scaffold N50 of de novo assemblies by eight- to 57-fold with high accuracy. We also demonstrate that fragScaff is complementary to Hi-C-based contact probability maps, providing midrange contiguity to support robust, accurate chromosome-scale de novo genome assemblies without the need for laborious in vivo cloning steps. Finally, we demonstrate CPT-seq as a means of anchoring unplaced novel human contigs to the reference genome as well as for detecting misassembled sequences.

  5. NxRepair: error correction in de novo sequence assembly using Nextera mate pairs.

    Science.gov (United States)

    Murphy, Rebecca R; O'Connell, Jared; Cox, Anthony J; Schulz-Trieglaff, Ole

    2015-01-01

    Scaffolding errors and incorrect repeat disambiguation during de novo assembly can result in large scale misassemblies in draft genomes. Nextera mate pair sequencing data provide additional information to resolve assembly ambiguities during scaffolding. Here, we introduce NxRepair, an open source toolkit for error correction in de novo assemblies that uses Nextera mate pair libraries to identify and correct large-scale errors. We show that NxRepair can identify and correct large scaffolding errors, without use of a reference sequence, resulting in quantitative improvements in the assembly quality. NxRepair can be downloaded from GitHub or PyPI, the Python Package Index; a tutorial and user documentation are also available.

  6. PRIME: A Mass Spectrum Data Mining Tool for De Novo Sequencing and PTMs Identification

    Institute of Scientific and Technical Information of China (English)

    Bo Yan; You-Xing Qu; Feng-Lou Mao; Victor N. Olman; Ying Xu

    2005-01-01

    De novo sequencing is one of the most promising proteomics techniques for identification of protein posttranslation modifications (PTMs) in studying protein regulations and functions. We have developed a computer tool PRIME for identification of b and y ions in tandem mass spectra, a key challenging problem in de novo sequencing. PRIME utilizes a feature that ions of the same and different types follow different mass-difference distributions to separate b from y ions correctly. We have formulated the problem as a graph partition problem. A linear integer-programming algorithm has been implemented to solve the graph partition problem rigorously and efficiently. The performance of PRIME has been demonstrated on a large amount of simulated tandem mass spectra derived from Yeast genome and its power of detecting PTMs has been tested on 216 simulated phosphopeptides.

  7. NxRepair: error correction in de novo sequence assembly using Nextera mate pairs

    Directory of Open Access Journals (Sweden)

    Rebecca R. Murphy

    2015-06-01

    Full Text Available Scaffolding errors and incorrect repeat disambiguation during de novo assembly can result in large scale misassemblies in draft genomes. Nextera mate pair sequencing data provide additional information to resolve assembly ambiguities during scaffolding. Here, we introduce NxRepair, an open source toolkit for error correction in de novo assemblies that uses Nextera mate pair libraries to identify and correct large-scale errors. We show that NxRepair can identify and correct large scaffolding errors, without use of a reference sequence, resulting in quantitative improvements in the assembly quality. NxRepair can be downloaded from GitHub or PyPI, the Python Package Index; a tutorial and user documentation are also available.

  8. De novo characterization of a whitefly transcriptome and analysis of its gene expression during development.

    Science.gov (United States)

    Wang, Xiao-Wei; Luan, Jun-Bo; Li, Jun-Min; Bao, Yan-Yuan; Zhang, Chuan-Xi; Liu, Shu-Sheng

    2010-06-24

    Whitefly (Bemisia tabaci) causes extensive crop damage throughout the world by feeding directly on plants and by vectoring hundreds of species of begomoviruses. Yet little is understood about its genes involved in development, insecticide resistance, host range plasticity and virus transmission. To facilitate research on whitefly, we present a method for de novo assembly of whitefly transcriptome using short read sequencing technology (Illumina). In a single run, we produced more than 43 million sequencing reads. These reads were assembled into 168,900 unique sequences (mean size = 266 bp) which represent more than 10-fold of all the whitefly sequences deposited in the GenBank (as of March 2010). Based on similarity search with known proteins, these analyses identified 27,290 sequences with a cut-off E-value above 10-5. Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. In addition, we investigated the transcriptome changes during whitefly development using a tag-based digital gene expression (DGE) system. We obtained a sequencing depth of over 2.5 million tags per sample and identified a large number of genes associated with specific developmental stages and insecticide resistance. Our data provides the most comprehensive sequence resource available for whitefly study and demonstrates that the Illumina sequencing allows de novo transcriptome assembly and gene expression analysis in a species lacking genome information. We anticipate that next generation sequencing technologies hold great potential for the study of the transcriptome in other non-model organisms.

  9. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences

    OpenAIRE

    Li, Heng

    2015-01-01

    Motivation: Single Molecule Real-Time (SMRT) sequencing technology and Oxford Nanopore technologies (ONT) produce reads over 10kbp in length, which have enabled high-quality genome assembly at an affordable cost. However, at present, long reads have an error rate as high as 10-15%. Complex and computationally intensive pipelines are required to assemble such reads. Results: We present a new mapper, minimap, and a de novo assembler, miniasm, for efficiently mapping and assembling SMRT and ONT ...

  10. De Novo Transcriptome Sequencing in Trigonella foenum-graecum L. to Identify Genes Involved in the Biosynthesis of Diosgenin

    Directory of Open Access Journals (Sweden)

    Kanak Vaidya

    2013-07-01

    Full Text Available T L. (fenugreek is a viable alternative for production of diosgenin because of its shorter growing cycle, lower production costs, and consistent yield and quality. We studied de novo transcriptome analysis along with the diosgenin pathway in and identified the genes responsible for diosgenin biosynthesis. The GMV-1 variety of has been used in the present study for transcriptome analysis by sequencing messenger ribonucleic acid (RNA with a SOLiD 4 Genome Analyzer. Deep sequencing data of the transcriptome was assembled using various assembly tools along with functional annotation of genes, and pathway analysis for diosgenin biosynthesis was deciphered. A total of 42 million high quality reads were obtained. De novo assembly was performed using Velvet at different -mer, Oases, and CLC Genomics Workbench, which generated 20,561 transcript contigs. CAP3 was used to reduce the redundancy of contigs obtained through these assemblers. A total of 18,333 transcript contigs were functionally annotated. Kyoto Encyclopedia of Genes and Genomes pathway mapping showed that 6775 transcripts were related to plant biochemical pathways including the diosgenin biosynthesis pathway. The large number of transcripts reported in the current study will serve as a valuable genetic resource for Sequence information of the genes that were involved in diosgenin biosynthesis could be used for metabolic engineering of to increase diosgenin content.

  11. Transcriptome Sequencing and De Novo Assembly of Golden Cuttlefish Sepia esculenta Hoyle

    Directory of Open Access Journals (Sweden)

    Changlin Liu

    2016-10-01

    Full Text Available Golden cuttlefish Sepia esculenta Hoyle is an economically important cephalopod species. However, artificial hatching is currently challenged by low survival rate of larvae due to abnormal embryonic development. Dissecting the genetic foundation and regulatory mechanisms in embryonic development requires genomic background knowledge. Therefore, we carried out a transcriptome sequencing on Sepia embryos and larvae via mRNA-Seq. 32,597,241 raw reads were filtered and assembled into 98,615 unigenes (N50 length at 911 bp which were annotated in NR database, GO and KEGG databases respectively. Digital gene expression analysis was carried out on cleavage stage embryos, healthy larvae and malformed larvae. Unigenes functioning in cell proliferation exhibited higher transcriptional levels at cleavage stage while those related to animal disease and organ development showed increased transcription in malformed larvae. Homologs of key genes in regulatory pathways related to early development of animals were identified in Sepia. Most of them exhibit higher transcriptional levels in cleavage stage than larvae, suggesting their potential roles in embryonic development of Sepia. The de novo assembly of Sepia transcriptome is fundamental genetic background for further exploration in Sepia research. Our demonstration on the transcriptional variations of genes in three developmental stages will provide new perspectives in understanding the molecular mechanisms in early embryonic development of cuttlefish.

  12. De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome

    Directory of Open Access Journals (Sweden)

    Iorizzo Massimo

    2012-05-01

    Full Text Available Abstract Background Sequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA. Results Sequencing data from a carrot 454 whole genome library were used to develop a de novo assembly of the mitochondrial genome. Development of a new bioinformatic tool allowed visualizing contig connections and elucidation of the de novo assembly. Southern hybridization demonstrated recombination across two large repeats. Genome annotation allowed identification of 44 protein coding genes, three rRNA and 17 tRNA. Identification of the plastid genome sequence allowed organelle genome comparison. Mitochondrial intergenic sequence analysis allowed detection of a fragment of DNA specific to the carrot plastid genome. PCR amplification and sequence analysis across different Apiaceae species revealed consistent conservation of this fragment in the mitochondrial genomes and an insertion in Daucus plastid genomes, giving evidence of a mitochondrial to plastid transfer of DNA. Sequence similarity with a retrotransposon element suggests a possibility that a transposon-like event transferred this sequence into the plastid genome. Conclusions This study confirmed that whole genome sequencing is a practical approach for de novo assembly of higher plant mitochondrial genomes. In addition, a new aspect of intercompartmental genome interaction was reported providing the first evidence for DNA transfer into an angiosperm plastid genome. The approach used here could be used more broadly to sequence and assemble mitochondrial genomes of diverse species. This information will allow us to better understand intercompartmental interactions and cell evolution.

  13. De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments

    Directory of Open Access Journals (Sweden)

    Gahlan Parul

    2012-03-01

    Full Text Available Abstract Background Picrorhiza kurrooa Royle ex Benth. is an endangered plant species of medicinal importance. The medicinal property is attributed to monoterpenoids picroside I and II, which are modulated by temperature. The transcriptome information of this species is limited with the availability of few hundreds of expressed sequence tags (ESTs in the public databases. In order to gain insight into temperature mediated molecular changes, high throughput de novo transcriptome sequencing and analyses were carried out at 15°C and 25°C, the temperatures known to modulate picrosides content. Results Using paired-end (PE Illumina sequencing technology, a total of 20,593,412 and 44,229,272 PE reads were obtained after quality filtering for 15°C and 25°C, respectively. Available (e.g., De-Bruijn/Eulerian graph and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 74,336 assembled transcript sequences were obtained, with an average coverage of 76.6 and average length of 439.5. Guanine-cytosine (GC content was observed to be 44.6%, while the transcriptome exhibited abundance of trinucleotide simple sequence repeat (SSR; 45.63% markers. Large scale expression profiling through "read per exon kilobase per million (RPKM", showed changes in several biological processes and metabolic pathways including cytochrome P450s (CYPs, UDP-glycosyltransferases (UGTs and those associated with picrosides biosynthesis. RPKM data were validated by reverse transcriptase-polymerase chain reaction using a set of 19 genes, wherein 11 genes behaved in accordance with the two expression methods. Conclusions Study generated transcriptome of P. kurrooa at two different temperatures. Large scale expression profiling through RPKM showed major transcriptome changes in response to temperature reflecting alterations in major biological processes and metabolic pathways, and provided insight of GC content and SSR markers

  14. Long-read sequencing and de novo assembly of a Chinese genome.

    Science.gov (United States)

    Shi, Lingling; Guo, Yunfei; Dong, Chengliang; Huddleston, John; Yang, Hui; Han, Xiaolu; Fu, Aisi; Li, Quan; Li, Na; Gong, Siyi; Lintner, Katherine E; Ding, Qiong; Wang, Zou; Hu, Jiang; Wang, Depeng; Wang, Feng; Wang, Lin; Lyon, Gholson J; Guan, Yongtao; Shen, Yufeng; Evgrafov, Oleg V; Knowles, James A; Thibaud-Nissen, Francoise; Schneider, Valerie; Yu, Chack-Yung; Zhou, Libing; Eichler, Evan E; So, Kwok-Fai; Wang, Kai

    2016-06-30

    Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arrays and generate a de novo assembly of 2.93 Gb (contig N50: 8.3 Mb, scaffold N50: 22.0 Mb, including 39.3 Mb N-bases), together with 206 Mb of alternative haplotypes. The assembly fully or partially fills 274 (28.4%) N-gaps in the reference genome GRCh38. Comparison to GRCh38 reveals 12.8 Mb of HX1-specific sequences, including 4.1 Mb that are not present in previously reported Asian genomes. Furthermore, long-read sequencing of the transcriptome reveals novel spliced genes that are not annotated in GENCODE and are missed by short-read RNA-Seq. Our results imply that improved characterization of genome functional variation may require the use of a range of genomic technologies on diverse human populations.

  15. De Novo Transcriptome Assembly of the Chinese Swamp Buffalo by RNA Sequencing and SSR Marker Discovery.

    Directory of Open Access Journals (Sweden)

    Tingxian Deng

    Full Text Available The Chinese swamp buffalo (Bubalis bubalis is vital to the lives of small farmers and has tremendous economic importance. However, a lack of genomic information has hampered research on augmenting marker assisted breeding programs in this species. Thus, a high-throughput transcriptomic sequencing of B. bubalis was conducted to generate transcriptomic sequence dataset for gene discovery and molecular marker development. Illumina paired-end sequencing generated a total of 54,109,173 raw reads. After trimming, de novo assembly was performed, which yielded 86,017 unigenes, with an average length of 972.41 bp, an N50 of 1,505 bp, and an average GC content of 49.92%. A total of 62,337 unigenes were successfully annotated. Among the annotated unigenes, 27,025 (43.35% and 23,232 (37.27% unigenes showed significant similarity to known proteins in NCBI non-redundant protein and Swiss-Prot databases (E-value < 1.0E-5, respectively. Of these annotated unigenes, 14,439 and 15,813 unigenes were assigned to the Gene Ontology (GO categories and EuKaryotic Ortholog Group (KOG cluster, respectively. In addition, a total of 14,167 unigenes were assigned to 331 Kyoto Encyclopedia of Genes and Genomes (KEGG pathways. Furthermore, 17,401 simple sequence repeats (SSRs were identified as potential molecular markers. One hundred and fifteen primer pairs were randomly selected for amplification to detect polymorphisms. The results revealed that 110 primer pairs (95.65% yielded PCR amplicons and 69 primer pairs (60.00% presented polymorphisms in 35 individual buffaloes. A phylogenetic analysis showed that the five swamp buffalo populations were clustered together, whereas two river buffalo breeds clustered separately. In the present study, the Illumina RNA-seq technology was utilized to perform transcriptome analysis and SSR marker discovery in the swamp buffalo without using a reference genome. Our findings will enrich the current SSR markers resources and help spearhead

  16. De novo Transcriptome Analysis in Perennial Ryegrass

    DEFF Research Database (Denmark)

    Farrell, Jacqueline Danielle; Byrne, Stephen; Asp, Torben

    heterozygous genotypes to enable SNP discovery for marker-assisted selection (MAS) and to determine the number of synonymous and non-synonymous SNPs. In this study we have preformed RNA-seq analysis of leaf, stem, inflorescence, leaf sheath, root and meristem from the inbred and heterozygous genotypes; to get...

  17. Annotation and Re-Sequencing of Genes from De Novo Transcriptome Assembly of Abies alba (Pinaceae

    Directory of Open Access Journals (Sweden)

    Anna M. Roschanski

    2013-01-01

    Full Text Available Premise of the study: We present a protocol for the annotation of transcriptome sequence data and the identification of candidate genes therein using the example of the nonmodel conifer Abies alba. Methods and Results: A normalized cDNA library was built from an A. alba seedling. The sequencing on a 454 platform yielded more than 1.5 million reads that were de novo assembled into 25 149 contigs. Two complementary approaches were applied to annotate gene fragments that code for (1 well-known proteins and (2 proteins that are potentially adaptively relevant. Primer development and testing yielded 88 amplicons that could successfully be resequenced from genomic DNA. Conclusions: The annotation workflow offers an efficient way to identify potential adaptively relevant genes from the large quantity of transcriptome sequence data. The primer set presented should be prioritized for single-nucleotide polymorphism detection in adaptively relevant genes in A. alba.

  18. The sequence and de novo assembly of the giant panda genome

    Science.gov (United States)

    Li, Ruiqiang; Fan, Wei; Tian, Geng; Zhu, Hongmei; He, Lin; Cai, Jing; Huang, Quanfei; Cai, Qingle; Li, Bo; Bai, Yinqi; Zhang, Zhihe; Zhang, Yaping; Wang, Wen; Li, Jun; Wei, Fuwen; Li, Heng; Jian, Min; Li, Jianwen; Zhang, Zhaolei; Nielsen, Rasmus; Li, Dawei; Gu, Wanjun; Yang, Zhentao; Xuan, Zhaoling; Ryder, Oliver A.; Leung, Frederick Chi-Ching; Zhou, Yan; Cao, Jianjun; Sun, Xiao; Fu, Yonggui; Fang, Xiaodong; Guo, Xiaosen; Wang, Bo; Hou, Rong; Shen, Fujun; Mu, Bo; Ni, Peixiang; Lin, Runmao; Qian, Wubin; Wang, Guodong; Yu, Chang; Nie, Wenhui; Wang, Jinhuan; Wu, Zhigang; Liang, Huiqing; Min, Jiumeng; Wu, Qi; Cheng, Shifeng; Ruan, Jue; Wang, Mingwei; Shi, Zhongbin; Wen, Ming; Liu, Binghang; Ren, Xiaoli; Zheng, Huisong; Dong, Dong; Cook, Kathleen; Shan, Gao; Zhang, Hao; Kosiol, Carolin; Xie, Xueying; Lu, Zuhong; Zheng, Hancheng; Li, Yingrui; Steiner, Cynthia C.; Lam, Tommy Tsan-Yuk; Lin, Siyuan; Zhang, Qinghui; Li, Guoqing; Tian, Jing; Gong, Timing; Liu, Hongde; Zhang, Dejin; Fang, Lin; Ye, Chen; Zhang, Juanbin; Hu, Wenbo; Xu, Anlong; Ren, Yuanyuan; Zhang, Guojie; Bruford, Michael W.; Li, Qibin; Ma, Lijia; Guo, Yiran; An, Na; Hu, Yujie; Zheng, Yang; Shi, Yongyong; Li, Zhiqiang; Liu, Qing; Chen, Yanling; Zhao, Jing; Qu, Ning; Zhao, Shancen; Tian, Feng; Wang, Xiaoling; Wang, Haiyin; Xu, Lizhi; Liu, Xiao; Vinar, Tomas; Wang, Yajun; Lam, Tak-Wah; Yiu, Siu-Ming; Liu, Shiping; Zhang, Hemin; Li, Desheng; Huang, Yan; Wang, Xia; Yang, Guohua; Jiang, Zhi; Wang, Junyi; Qin, Nan; Li, Li; Li, Jingxiang; Bolund, Lars; Kristiansen, Karsten; Wong, Gane Ka-Shu; Olson, Maynard; Zhang, Xiuqing; Li, Songgang; Yang, Huanming; Wang, Jian; Wang, Jun

    2013-01-01

    Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes. PMID:20010809

  19. De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

    Science.gov (United States)

    Nowrousian, Minou; Stajich, Jason E; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D; Pöggeler, Stefanie; Read, Nick D; Seiler, Stephan; Smith, Kristina M; Zickler, Denise; Kück, Ulrich; Freitag, Michael

    2010-04-08

    Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for

  20. Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

    Directory of Open Access Journals (Sweden)

    Madlung Johannes

    2010-05-01

    Full Text Available Abstract Background Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i. Results The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set. All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase. Conclusions Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

  1. Antilope - A Lagrangian Relaxation Approach to the de novo Peptide Sequencing Problem

    CERN Document Server

    Andreotti, Sandro; Reinert, Knut

    2011-01-01

    Peptide sequencing from mass spectrometry data is a key step in proteome research. Especially de novo sequencing, the identification of a peptide from its spectrum alone, is still a challenge even for state-of-the-art algorithmic approaches. In this paper we present Antilope, a new fast and flexible approach based on mathematical programming. It builds on the spectrum graph model and works with a variety of scoring schemes. Antilope combines Lagrangian relaxation for solving an integer linear programming formulation with an adaptation of Yen's k shortest paths algorithm. It shows a significant improvement in running time compared to mixed integer optimization and performs at the same speed like other state-of-the-art tools. We also implemented a generic probabilistic scoring scheme that can be trained automatically for a dataset of annotated spectra and is independent of the mass spectrometer type. Evaluations on benchmark data show that Antilope is competitive to the popular state-of-the-art programs PepNovo...

  2. Evaluation of methods for de novo genome assembly from high-throughput sequencing reads reveals dependencies that affect the quality of the results.

    Science.gov (United States)

    Haiminen, Niina; Kuhn, David N; Parida, Laxmi; Rigoutsos, Isidore

    2011-01-01

    Recent developments in high-throughput sequencing technology have made low-cost sequencing an attractive approach for many genome analysis tasks. Increasing read lengths, improving quality and the production of increasingly larger numbers of usable sequences per instrument-run continue to make whole-genome assembly an appealing target application. In this paper we evaluate the feasibility of de novo genome assembly from short reads (≤100 nucleotides) through a detailed study involving genomic sequences of various lengths and origin, in conjunction with several of the currently popular assembly programs. Our extensive analysis demonstrates that, in addition to sequencing coverage, attributes such as the architecture of the target genome, the identity of the used assembly program, the average read length and the observed sequencing error rates are powerful variables that affect the best achievable assembly of the target sequence in terms of size and correctness.

  3. A Real-Time de novo DNA Sequencing Assembly Platform Based on an FPGA Implementation.

    Science.gov (United States)

    Hu, Yuanqi; Georgiou, Pantelis

    2016-01-01

    This paper presents an FPGA based DNA comparison platform which can be run concurrently with the sensing phase of DNA sequencing and shortens the overall time needed for de novo DNA assembly. A hybrid overlap searching algorithm is applied which is scalable and can deal with incremental detection of new bases. To handle the incomplete data set which gradually increases during sequencing time, all-against-all comparisons are broken down into successive window-against-window comparison phases and executed using a novel dynamic suffix comparison algorithm combined with a partitioned dynamic programming method. The complete system has been designed to facilitate parallel processing in hardware, which allows real-time comparison and full scalability as well as a decrease in the number of computations required. A base pair comparison rate of 51.2 G/s is achieved when implemented on an FPGA with successful DNA comparison when using data sets from real genomes.

  4. De Novo Transcriptome Sequencing of Desert Herbaceous Achnatherum splendens (Achnatherum Seedlings and Identification of Salt Tolerance Genes

    Directory of Open Access Journals (Sweden)

    Jiangtao Liu

    2016-03-01

    Full Text Available Achnatherum splendens is an important forage herb in Northwestern China. It has a high tolerance to salinity and is, thus, considered one of the most important constructive plants in saline and alkaline areas of land in Northwest China. However, the mechanisms of salt stress tolerance in A. splendens remain unknown. Next-generation sequencing (NGS technologies can be used for global gene expression profiling. In this study, we examined sequence and transcript abundance data for the root/leaf transcriptome of A. splendens obtained using an Illumina HiSeq 2500. Over 35 million clean reads were obtained from the leaf and root libraries. All of the RNA sequencing (RNA-seq reads were assembled de novo into a total of 126,235 unigenes and 36,511 coding DNA sequences (CDS. We further identified 1663 differentially-expressed genes (DEGs between the salt stress treatment and control. Functional annotation of the DEGs by gene ontology (GO, using Arabidopsis and rice as references, revealed enrichment of salt stress-related GO categories, including “oxidation reduction”, “transcription factor activity”, and “ion channel transporter”. Thus, this global transcriptome analysis of A. splendens has provided an important genetic resource for the study of salt tolerance in this halophyte. The identified sequences and their putative functional data will facilitate future investigations of the tolerance of Achnatherum species to various types of abiotic stress.

  5. De novo sequence assembly and characterization of Lycoris aurea transcriptome using GS FLX titanium platform of 454 pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Ren Wang

    Full Text Available BACKGROUND: Lycoris aurea, also called Golden Magic Lily, is an ornamentally and medicinally important species of the Amaryllidaceae family. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST dataset for L. aurea using high-throughput sequencing technology. METHODOLOGY AND PRINCIPAL FINDINGS: Total RNA was isolated from leaves with sodium nitroprusside (SNP, salicylic acid (SA, or methyl jasmonate (MeJA treatment, stems, and flowers at the bud, blooming, and wilting stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, a total of 937,990 high quality reads (308.63 Mb with an average read length of 329 bp were generated. Clustering and assembly of these reads produced a non-redundant set of 141,111 unique sequences, comprising 24,604 contigs and 116,507 singletons. All of the unique sequences were involved in the biological process, cellular component and molecular function categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literatures, many putative genes involved in Amaryllidaceae alkaloids synthesis, including PAL, TYDC OMT, NMT, P450, and other potentially important candidate genes, were identified for the first time in this Lycoris. Furthermore, 6,386 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset. CONCLUSIONS: The transcriptome provides an invaluable new data for a functional genomics resource and future biological research in L. aurea. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will provide useful

  6. Fine de novo sequencing of a fungal genome using only SOLiD short read data: verification on Aspergillus oryzae RIB40.

    Directory of Open Access Journals (Sweden)

    Myco Umemura

    Full Text Available The development of next-generation sequencing (NGS technologies has dramatically increased the throughput, speed, and efficiency of genome sequencing. The short read data generated from NGS platforms, such as SOLiD and Illumina, are quite useful for mapping analysis. However, the SOLiD read data with lengths of <60 bp have been considered to be too short for de novo genome sequencing. Here, to investigate whether de novo sequencing of fungal genomes is possible using only SOLiD short read sequence data, we performed de novo assembly of the Aspergillus oryzae RIB40 genome using only SOLiD read data of 50 bp generated from mate-paired libraries with 2.8- or 1.9-kb insert sizes. The assembled scaffolds showed an N50 value of 1.6 Mb, a 22-fold increase than those obtained using only SOLiD short read in other published reports. In addition, almost 99% of the reference genome was accurately aligned by the assembled scaffold fragments in long lengths. The sequences of secondary metabolite biosynthetic genes and clusters, whose products are of considerable interest in fungal studies due to their potential medicinal, agricultural, and cosmetic properties, were also highly reconstructed in the assembled scaffolds. Based on these findings, we concluded that de novo genome sequencing using only SOLiD short reads is feasible and practical for molecular biological study of fungi. We also investigated the effect of filtering low quality data, library insert size, and k-mer size on the assembly performance, and recommend for the assembly use of mild filtered read data where the N50 was not so degraded and the library has an insert size of ∼2.0 kb, and k-mer size 33.

  7. Identifying wrong assemblies in de novo short read primary sequence assembly contigs

    Indian Academy of Sciences (India)

    VANDNA CHAWLA; RAJNISH KUMAR; RAVI SHANKAR

    2016-09-01

    With the advent of short-reads-based genome sequencing approaches, large number of organisms are being sequencedall over the world. Most of these assemblies are done using some de novo short read assemblers and other relatedapproaches. However, the contigs produced this way are prone to wrong assembly. So far, there is a conspicuousdearth of reliable tools to identify mis-assembled contigs. Mis-assemblies could result from incorrectly deleted orwrongly arranged genomic sequences. In the present work various factors related to sequence, sequencing andassembling have been assessed for their role in causing mis-assembly by using different genome sequencing data.Finally, some mis-assembly detecting tools have been evaluated for their ability to detect the wrongly assembledprimary contigs, suggesting a lot of scope for improvement in this area. The present work also proposes a simpleunsupervised learning-based novel approach to identify mis-assemblies in the contigs which was found performingreasonably well when compared to the already existing tools to report mis-assembled contigs. It was observed that theproposed methodology may work as a complementary system to the existing tools to enhance their accuracy.

  8. De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa.

    Science.gov (United States)

    Kim, Hyun A; Shin, Ah-Young; Lee, Min-Seon; Lee, Hee-Jeong; Lee, Heung-Ryul; Ahn, Jongmoon; Nahm, Seokhyeon; Jo, Sung-Hwan; Park, Jeong Mee; Kwon, Suk-Yoon

    2016-02-01

    Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family.

  9. De novo assembly, gene annotation and marker development using Illumina paired-end transcriptome sequences in celery (Apium graveolens L..

    Directory of Open Access Journals (Sweden)

    Nan Fu

    Full Text Available BACKGROUND: Celery is an increasing popular vegetable species, but limited transcriptome and genomic data hinder the research to it. In addition, a lack of celery molecular markers limits the process of molecular genetic breeding. High-throughput transcriptome sequencing is an efficient method to generate a large transcriptome sequence dataset for gene discovery, molecular marker development and marker-assisted selection breeding. PRINCIPAL FINDINGS: Celery transcriptomes from four tissues were sequenced using Illumina paired-end sequencing technology. De novo assembling was performed to generate a collection of 42,280 unigenes (average length of 502.6 bp that represent the first transcriptome of the species. 78.43% and 48.93% of the unigenes had significant similarity with proteins in the National Center for Biotechnology Information (NCBI non-redundant protein database (Nr and Swiss-Prot database respectively, and 10,473 (24.77% unigenes were assigned to Clusters of Orthologous Groups (COG. 21,126 (49.97% unigenes harboring Interpro domains were annotated, in which 15,409 (36.45% were assigned to Gene Ontology(GO categories. Additionally, 7,478 unigenes were mapped onto 228 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG. Large numbers of simple sequence repeats (SSRs were indentified, and then the rate of successful amplication and polymorphism were investigated among 31 celery accessions. CONCLUSIONS: This study demonstrates the feasibility of generating a large scale of sequence information by Illumina paired-end sequencing and efficient assembling. Our results provide a valuable resource for celery research. The developed molecular markers are the foundation of further genetic linkage analysis and gene localization, and they will be essential to accelerate the process of breeding.

  10. De Novo Transcriptome Sequencing of Oryza officinalis Wall ex Watt to Identify Disease-Resistance Genes

    Directory of Open Access Journals (Sweden)

    Bin He

    2015-12-01

    Full Text Available Oryza officinalis Wall ex Watt is one of the most important wild relatives of cultivated rice and exhibits high resistance to many diseases. It has been used as a source of genes for introgression into cultivated rice. However, there are limited genomic resources and little genetic information publicly reported for this species. To better understand the pathways and factors involved in disease resistance and accelerating the process of rice breeding, we carried out a de novo transcriptome sequencing of O. officinalis. In this research, 137,229 contigs were obtained ranging from 200 to 19,214 bp with an N50 of 2331 bp through de novo assembly of leaves, stems and roots in O. officinalis using an Illumina HiSeq 2000 platform. Based on sequence similarity searches against a non-redundant protein database, a total of 88,249 contigs were annotated with gene descriptions and 75,589 transcripts were further assigned to GO terms. Candidate genes for plant–pathogen interaction and plant hormones regulation pathways involved in disease-resistance were identified. Further analyses of gene expression profiles showed that the majority of genes related to disease resistance were all expressed in the three tissues. In addition, there are two kinds of rice bacterial blight-resistant genes in O. officinalis, including two Xa1 genes and three Xa26 genes. All 2 Xa1 genes showed the highest expression level in stem, whereas one of Xa26 was expressed dominantly in leaf and other 2 Xa26 genes displayed low expression level in all three tissues. This transcriptomic database provides an opportunity for identifying the genes involved in disease-resistance and will provide a basis for studying functional genomics of O. officinalis and genetic improvement of cultivated rice in the future.

  11. Classifying Genomic Sequences by Sequence Feature Analysis

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua Liu; Dian Jiao; Xiao Sun

    2005-01-01

    Traditional sequence analysis depends on sequence alignment. In this study, we analyzed various functional regions of the human genome based on sequence features, including word frequency, dinucleotide relative abundance, and base-base correlation. We analyzed the human chromosome 22 and classified the upstream,exon, intron, downstream, and intergenic regions by principal component analysis and discriminant analysis of these features. The results show that we could classify the functional regions of genome based on sequence feature and discriminant analysis.

  12. De novo analysis of transcriptome dynamics in the migratory locust during the development of phase traits.

    Directory of Open Access Journals (Sweden)

    Shuang Chen

    Full Text Available Locusts exhibit remarkable density-dependent phenotype (phase changes from the solitary to the gregarious, making them one of the most destructive agricultural pests. This phenotype polyphenism arises from a single genome and diverse transcriptomes in different conditions. Here we report a de novo transcriptome for the migratory locust and a comprehensive, representative core gene set. We carried out assembly of 21.5 Gb Illumina reads, generated 72,977 transcripts with N50 2,275 bp and identified 11,490 locust protein-coding genes. Comparative genomics analysis with eight other sequenced insects was carried out to identify the genomic divergence between hemimetabolous and holometabolous insects for the first time and 18 genes relevant to development was found. We further utilized the quantitative feature of RNA-seq to measure and compare gene expression among libraries. We first discovered how divergence in gene expression between two phases progresses as locusts develop and identified 242 transcripts as candidates for phase marker genes. Together with the detailed analysis of deep sequencing data of the 4(th instar, we discovered a phase-dependent divergence of biological investment in the molecular level. Solitary locusts have higher activity in biosynthetic pathways while gregarious locusts show higher activity in environmental interaction, in which genes and pathways associated with regulation of neurotransmitter activities, such as neurotransmitter receptors, synthetase, transporters, and GPCR signaling pathways, are strongly involved. Our study, as the largest de novo transcriptome to date, with optimization of sequencing and assembly strategy, can further facilitate the application of de novo transcriptome. The locust transcriptome enriches genetic resources for hemimetabolous insects and our understanding of the origin of insect metamorphosis. Most importantly, we identified genes and pathways that might be involved in locust development

  13. Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome.

    Science.gov (United States)

    Goodwin, Sara; Gurtowski, James; Ethe-Sayers, Scott; Deshpande, Panchajanya; Schatz, Michael C; McCombie, W Richard

    2015-11-01

    Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades. Recently, a nanopore-based sequencing instrument, the Oxford Nanopore MinION, has become available, and we used this for sequencing the Saccharomyces cerevisiae genome. To make use of these data, we developed a novel open-source hybrid error correction algorithm Nanocorr specifically for Oxford Nanopore reads, because existing packages were incapable of assembling the long read lengths (5-50 kbp) at such high error rates (between ∼5% and 40% error). With this new method, we were able to perform a hybrid error correction of the nanopore reads using complementary MiSeq data and produce a de novo assembly that is highly contiguous and accurate: The contig N50 length is more than ten times greater than an Illumina-only assembly (678 kb versus 59.9 kbp) and has >99.88% consensus identity when compared to the reference. Furthermore, the assembly with the long nanopore reads presents a much more complete representation of the features of the genome and correctly assembles gene cassettes, rRNAs, transposable elements, and other genomic features that were almost entirely absent in the Illumina-only assembly.

  14. Biological sequence analysis

    DEFF Research Database (Denmark)

    Durbin, Richard; Eddy, Sean; Krogh, Anders Stærmose

    This book provides an up-to-date and tutorial-level overview of sequence analysis methods, with particular emphasis on probabilistic modelling. Discussed methods include pairwise alignment, hidden Markov models, multiple alignment, profile searches, RNA secondary structure analysis, and phylogene......This book provides an up-to-date and tutorial-level overview of sequence analysis methods, with particular emphasis on probabilistic modelling. Discussed methods include pairwise alignment, hidden Markov models, multiple alignment, profile searches, RNA secondary structure analysis...

  15. De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube.

    Science.gov (United States)

    Iaria, Domenico; Chiappetta, Adriana; Muzzalupo, Innocenzo

    2016-01-01

    In olive (Olea europaea L.), the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cultivars, have a distinct gene expression profile and many of the differentially expressed sequences between the samples fall within gene families involved in the development of the pollen tube, such as lipase, carboxylesterase, pectinesterase, pectin methylesterase, and callose synthase. Moreover, different genes involved in signal transduction, transcription, and growth are overrepresented. The analysis also allowed us to identify members in actin and actin depolymerization factor and fibrin gene family and member of the Ca(2+) binding gene family related to the development and polarization of pollen apical tip. The whole transcriptomic analysis, through the identification of the differentially expressed transcripts set and an extended functional annotation analysis, will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth in the olive.

  16. De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube

    Directory of Open Access Journals (Sweden)

    Domenico Iaria

    2016-01-01

    Full Text Available In olive (Olea europaea L., the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cultivars, have a distinct gene expression profile and many of the differentially expressed sequences between the samples fall within gene families involved in the development of the pollen tube, such as lipase, carboxylesterase, pectinesterase, pectin methylesterase, and callose synthase. Moreover, different genes involved in signal transduction, transcription, and growth are overrepresented. The analysis also allowed us to identify members in actin and actin depolymerization factor and fibrin gene family and member of the Ca2+ binding gene family related to the development and polarization of pollen apical tip. The whole transcriptomic analysis, through the identification of the differentially expressed transcripts set and an extended functional annotation analysis, will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth in the olive.

  17. Optimizing information in Next-Generation-Sequencing (NGS) reads for improving de novo genome assembly.

    Science.gov (United States)

    Liu, Tsunglin; Tsai, Cheng-Hung; Lee, Wen-Bin; Chiang, Jung-Hsien

    2013-01-01

    Next-Generation-Sequencing is advantageous because of its much higher data throughput and much lower cost compared with the traditional Sanger method. However, NGS reads are shorter than Sanger reads, making de novo genome assembly very challenging. Because genome assembly is essential for all downstream biological studies, great efforts have been made to enhance the completeness of genome assembly, which requires the presence of long reads or long distance information. To improve de novo genome assembly, we develop a computational program, ARF-PE, to increase the length of Illumina reads. ARF-PE takes as input Illumina paired-end (PE) reads and recovers the original DNA fragments from which two ends the paired reads are obtained. On the PE data of four bacteria, ARF-PE recovered >87% of the DNA fragments and achieved >98% of perfect DNA fragment recovery. Using Velvet, SOAPdenovo, Newbler, and CABOG, we evaluated the benefits of recovered DNA fragments to genome assembly. For all four bacteria, the recovered DNA fragments increased the assembly contiguity. For example, the N50 lengths of the P. brasiliensis contigs assembled by SOAPdenovo and Newbler increased from 80,524 bp to 166,573 bp and from 80,655 bp to 193,388 bp, respectively. ARF-PE also increased assembly accuracy in many cases. On the PE data of two fungi and a human chromosome, ARF-PE doubled and tripled the N50 length. However, the assembly accuracies dropped, but still remained >91%. In general, ARF-PE can increase both assembly contiguity and accuracy for bacterial genomes. For complex eukaryotic genomes, ARF-PE is promising because it raises assembly contiguity. But future error correction is needed for ARF-PE to also increase the assembly accuracy. ARF-PE is freely available at http://140.116.235.124/~tliu/arf-pe/.

  18. Sequencing and de novo transcriptome assembly of the Chinese giant salamander (Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Yong Huang

    2017-06-01

    Full Text Available Next-generation technologies for determination of genomics and transcriptomics composition have a wide range of applications. Andrias davidianus, has become an endangered amphibian species of salamander endemic in China. However, there is a lack of the molecular information. In this study, we obtained the RNA-Seq data from a pool of A. davidianus tissue including spleen, liver, muscle, kidney, skin, testis, gut and heart using Illumina HiSeq 2500 platform. A total of 15,398,997,600 bp were obtained, corresponding to 102,659,984 raw reads. A total of 102,659,984 reads were filtered after removing low-quality reads and trimming the adapter sequences. The Trinity program was used to de novo assemble 132,912 unigenes with an average length of 690 bp and N50 of 1263 bp. Unigenes were annotated through number of databases. These transcriptomic data of A. davidianus should open the door to molecular evolution studies based on the entire transcriptome or targeted genes of interest to sequence. The raw data in this study can be available in NCBI SRA database with accession number of SRP099564.

  19. Partial De Novo Sequencing and Unusual CID Fragmentation of a 7 kDa, Disulfide-Bridged Toxin

    Science.gov (United States)

    Medzihradszky, Katalin F.; Bohlen, Christopher J.

    2012-05-01

    A 7 kDa toxin isolated from the venom of the Texas coral snake ( Micrurus tener tener) was subjected to collision-induced dissociation (CID) and electron-transfer dissociation (ETD) analyses both before and after reduction at low pH. Manual and automated approaches to de novo sequencing are compared in detail. Manual de novo sequencing utilizing the combination of high accuracy CID and ETD data and an acid-related cleavage yielded the N-terminal half of the sequence from the reduced species. The intact polypeptide, containing 3 disulfide bridges produced a series of unusual fragments in ion trap CID experiments: abundant internal amino acid losses were detected, and also one of the disulfide-linkage positions could be determined from fragments formed by the cleavage of two bonds. In addition, internal and c-type fragments were also observed.

  20. Identification of lignin genes and regulatory sequences involved in secondary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing.

    Science.gov (United States)

    Wong, Melissa M L; Cannon, Charles H; Wickneswari, Ratnam

    2011-07-05

    Acacia auriculiformis × Acacia mangium hybrids are commercially important trees for the timber and pulp industry in Southeast Asia. Increasing pulp yield while reducing pulping costs are major objectives of tree breeding programs. The general monolignol biosynthesis and secondary cell wall formation pathways are well-characterized but genes in these pathways are poorly characterized in Acacia hybrids. RNA-seq on short-read platforms is a rapid approach for obtaining comprehensive transcriptomic data and to discover informative sequence variants. We sequenced transcriptomes of A. auriculiformis and A. mangium from non-normalized cDNA libraries synthesized from pooled young stem and inner bark tissues using paired-end libraries and a single lane of an Illumina GAII machine. De novo assembly produced a total of 42,217 and 35,759 contigs with an average length of 496 bp and 498 bp for A. auriculiformis and A. mangium respectively. The assemblies of A. auriculiformis and A. mangium had a total length of 21,022,649 bp and 17,838,260 bp, respectively, with the largest contig 15,262 bp long. We detected all ten monolignol biosynthetic genes using Blastx and further analysis revealed 18 lignin isoforms for each species. We also identified five contigs homologous to R2R3-MYB proteins in other plant species that are involved in transcriptional regulation of secondary cell wall formation and lignin deposition. We searched the contigs against public microRNA database and predicted the stem-loop structures of six highly conserved microRNA families (miR319, miR396, miR160, miR172, miR162 and miR168) and one legume-specific family (miR2086). Three microRNA target genes were predicted to be involved in wood formation and flavonoid biosynthesis. By using the assemblies as a reference, we discovered 16,648 and 9,335 high quality putative Single Nucleotide Polymorphisms (SNPs) in the transcriptomes of A. auriculiformis and A. mangium, respectively, thus yielding useful markers for

  1. Identification of lignin genes and regulatory sequences involved in secondary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing

    Directory of Open Access Journals (Sweden)

    Cannon Charles H

    2011-07-01

    Full Text Available Abstract Background Acacia auriculiformis × Acacia mangium hybrids are commercially important trees for the timber and pulp industry in Southeast Asia. Increasing pulp yield while reducing pulping costs are major objectives of tree breeding programs. The general monolignol biosynthesis and secondary cell wall formation pathways are well-characterized but genes in these pathways are poorly characterized in Acacia hybrids. RNA-seq on short-read platforms is a rapid approach for obtaining comprehensive transcriptomic data and to discover informative sequence variants. Results We sequenced transcriptomes of A. auriculiformis and A. mangium from non-normalized cDNA libraries synthesized from pooled young stem and inner bark tissues using paired-end libraries and a single lane of an Illumina GAII machine. De novo assembly produced a total of 42,217 and 35,759 contigs with an average length of 496 bp and 498 bp for A. auriculiformis and A. mangium respectively. The assemblies of A. auriculiformis and A. mangium had a total length of 21,022,649 bp and 17,838,260 bp, respectively, with the largest contig 15,262 bp long. We detected all ten monolignol biosynthetic genes using Blastx and further analysis revealed 18 lignin isoforms for each species. We also identified five contigs homologous to R2R3-MYB proteins in other plant species that are involved in transcriptional regulation of secondary cell wall formation and lignin deposition. We searched the contigs against public microRNA database and predicted the stem-loop structures of six highly conserved microRNA families (miR319, miR396, miR160, miR172, miR162 and miR168 and one legume-specific family (miR2086. Three microRNA target genes were predicted to be involved in wood formation and flavonoid biosynthesis. By using the assemblies as a reference, we discovered 16,648 and 9,335 high quality putative Single Nucleotide Polymorphisms (SNPs in the transcriptomes of A. auriculiformis and A. mangium

  2. De novo characterization of the Dialeurodes citri transcriptome: mining genes involved in stress resistance and simple sequence repeats (SSRs) discovery.

    Science.gov (United States)

    Chen, E-H; Wei, D-D; Shen, G-M; Yuan, G-R; Bai, P-P; Wang, J-J

    2014-02-01

    The citrus whitefly, Dialeurodes citri (Ashmead), is one of the three economically important whitefly species that infest citrus plants around the world; however, limited genetic research has been focused on D. citri, partly because of lack of genomic resources. In this study, we performed de novo assembly of a transcriptome using Illumina paired-end sequencing technology (Illumina Inc., San Diego, CA, USA). In total, 36,766 unigenes with a mean length of 497 bp were identified. Of these unigenes, we identified 17,788 matched known proteins in the National Center for Biotechnology Information database, as determined by Blast search, with 5731, 4850 and 14,441 unigenes assigned to clusters of orthologous groups (COG), gene ontology (GO), and SwissProt, respectively. In total, 7507 unigenes were assigned to 308 known pathways. In-depth analysis of the data showed that 117 unigenes were identified as potentially involved in the detoxification of xenobiotics and 67 heat shock protein (Hsp) genes were associated with environmental stress. In addition, these enzymes were searched against the GO and COG database, and the results showed that the three major detoxification enzymes and Hsps were classified into 18 and 3, 6, and 8 annotations, respectively. In addition, 149 simple sequence repeats were detected. The results facilitate the investigation of molecular resistance mechanisms to insecticides and environmental stress, and contribute to molecular marker development. The findings greatly improve our genetic understanding of D. citri, and lay the foundation for future functional genomics studies on this species.

  3. De novo transcriptome sequencing reveals a considerable bias in the incidence of simple sequence repeats towards the downstream of 'Pre-miRNAs' of black pepper.

    Directory of Open Access Journals (Sweden)

    Nisha Joy

    Full Text Available Next generation sequencing has an advantageon transformational development of species with limited available sequence data as it helps to decode the genome and transcriptome. We carried out the de novo sequencing using illuminaHiSeq™ 2000 to generate the first leaf transcriptome of black pepper (Piper nigrum L., an important spice variety native to South India and also grown in other tropical regions. Despite the economic and biochemical importance of pepper, a scientifically rigorous study at the molecular level is far from complete due to lack of sufficient sequence information and cytological complexity of its genome. The 55 million raw reads obtained, when assembled using Trinity program generated 2,23,386 contigs and 1,28,157 unigenes. Reports suggest that the repeat-rich genomic regions give rise to small non-coding functional RNAs. MicroRNAs (miRNAs are the most abundant type of non-coding regulatory RNAs. In spite of the widespread research on miRNAs, little is known about the hair-pin precursors of miRNAs bearing Simple Sequence Repeats (SSRs. We used the array of transcripts generated, for the in silico prediction and detection of '43 pre-miRNA candidates bearing different types of SSR motifs'. The analysis identified 3913 different types of SSR motifs with an average of one SSR per 3.04 MB of thetranscriptome. About 0.033% of the transcriptome constituted 'pre-miRNA candidates bearing SSRs'. The abundance, type and distribution of SSR motifs studied across the hair-pin miRNA precursors, showed a significant bias in the position of SSRs towards the downstream of predicted 'pre-miRNA candidates'. The catalogue of transcripts identified, together with the demonstration of reliable existence of SSRs in the miRNA precursors, permits future opportunities for understanding the genetic mechanism of black pepper and likely functions of 'tandem repeats' in miRNAs.

  4. Biological sequence analysis

    DEFF Research Database (Denmark)

    Durbin, Richard; Eddy, Sean; Krogh, Anders Stærmose

    This book provides an up-to-date and tutorial-level overview of sequence analysis methods, with particular emphasis on probabilistic modelling. Discussed methods include pairwise alignment, hidden Markov models, multiple alignment, profile searches, RNA secondary structure analysis, and phylogene...

  5. A biologist's guide to de novo genome assembly using next-generation sequence data: A test with fungal genomes.

    Science.gov (United States)

    Haridas, Sajeet; Breuill, Colette; Bohlmann, Joerg; Hsiang, Tom

    2011-09-01

    We offer a guide to de novo genome assembly using sequence data generated by the Illumina platform for biologists working with fungi or other organisms whose genomes are less than 100Mb in size. The guide requires no familiarity with sequencing assembly technology or associated computer programs. It defines commonly used terms in genome sequencing and assembly; provides examples of assembling short-read genome sequence data for four strains of the fungus Grosmannia clavigera using four assembly programs; gives examples of protocols and software; and presents a commented flowchart that extends from DNA preparation for submission to a sequencing center, through to processing and assembly of the raw sequence reads using freely available operating systems and software.

  6. De Novo Transcriptome Sequencing of Olea europaea L. to Identify Genes Involved in the Development of the Pollen Tube

    OpenAIRE

    Domenico Iaria; Adriana Chiappetta; Innocenzo Muzzalupo

    2016-01-01

    In olive (Olea europaea L.), the processes controlling self-incompatibility are still unclear and the molecular basis underlying this process are still not fully characterized. In order to determine compatibility relationships, using next-generation sequencing techniques and a de novo transcriptome assembly strategy, we show that pollen tubes from different olive plants, grown in vitro in a medium containing its own pistil and in combination pollen/pistil from self-sterile and self-fertile cu...

  7. De novo Assembly and Characterization of Cajanus scarabaeoides (L. Thouars Transcriptome by Paired-End Sequencing

    Directory of Open Access Journals (Sweden)

    Deepti Nigam

    2017-07-01

    Full Text Available Pigeonpea [Cajanus cajan (L. Millsp.] is a heat and drought resilient legume crop grown mostly in Asia and Africa. Pigeonpea is affected by various biotic (diseases and insect pests and abiotic stresses (salinity and water logging which limit the yield potential of this crop. However, resistance to all these constraints is not readily available in the cultivated genotypes and some of the wild relatives have been found to withstand these resistances. Thus, the utilization of crop wild relatives (CWR in pigeonpea breeding has been effective in conferring resistance, quality and breeding efficiency traits to this crop. Bud and leaf tissue of Cajanus scarabaeoides, a wild relative of pigeon pea were used for transcriptome profiling. Approximately 30 million clean reads filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (3.02 gigabase pairs were generated by Illumina paired-end RNA-seq technology. All of these clean reads were pooled and assembled de novo into 1,17,007 transcripts using the Trinity. Finally, a total of 98,664 unigenes were derived with mean length of 396 bp and N50 values of 1393. The assembly produced significant mapping results (73.68% in BLASTN searches of the Glycine max CDS sequence database (Ensembl. Further, uniprot database of Viridiplantae was used for unigene annotation; 81,799 of 98,664 (82.90% unigenes were finally annotated with gene descriptions or conserved protein domains. Further, a total of 23,475 SSRs were identified in 27,321 unigenes. This data will provide useful information for mining of functionally important genes and SSR markers for pigeonpea improvement.

  8. De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping.

    Science.gov (United States)

    Olsen, Remi-Andre; Bunikis, Ignas; Tiukova, Ievgeniia; Holmberg, Kicki; Lötstedt, Britta; Pettersson, Olga Vinnere; Passoth, Volkmar; Käller, Max; Vezzi, Francesco

    2015-01-01

    It remains a challenge to perform de novo assembly using next-generation sequencing (NGS). Despite the availability of multiple sequencing technologies and tools (e.g., assemblers) it is still difficult to assemble new genomes at chromosome resolution (i.e., one sequence per chromosome). Obtaining high quality draft assemblies is extremely important in the case of yeast genomes to better characterise major events in their evolutionary history. The aim of this work is two-fold: on the one hand we want to show how combining different and somewhat complementary technologies is key to improving assembly quality and correctness, and on the other hand we present a de novo assembly pipeline we believe to be beneficial to core facility bioinformaticians. To demonstrate both the effectiveness of combining technologies and the simplicity of the pipeline, here we present the results obtained using the Dekkera bruxellensis genome. In this work we used short-read Illumina data and long-read PacBio data combined with the extreme long-range information from OpGen optical maps in the task of de novo genome assembly and finishing. Moreover, we developed NouGAT, a semi-automated pipeline for read-preprocessing, de novo assembly and assembly evaluation, which was instrumental for this work. We obtained a high quality draft assembly of a yeast genome, resolved on a chromosomal level. Furthermore, this assembly was corrected for mis-assembly errors as demonstrated by resolving a large collapsed repeat and by receiving higher scores by assembly evaluation tools. With the inclusion of PacBio data we were able to fill about 5 % of the optical mapped genome not covered by the Illumina data.

  9. High throughput de novo RNA sequencing elucidates novel responses in Penicillium chrysogenum under microgravity.

    Science.gov (United States)

    Sathishkumar, Yesupatham; Krishnaraj, Chandran; Rajagopal, Kalyanaraman; Sen, Dwaipayan; Lee, Yang Soo

    2016-02-01

    In this study, the transcriptional alterations in Penicillium chrysogenum under simulated microgravity conditions were analyzed for the first time using an RNA-Seq method. The increasing plethora of eukaryotic microbial flora inside the spaceship demands the basic understanding of fungal biology in the absence of gravity vector. Penicillium species are second most dominant fungal contaminant in International Space Station. Penicillium chrysogenum an industrially important organism also has the potential to emerge as an opportunistic pathogen for the astronauts during the long-term space missions. But till date, the cellular mechanisms underlying the survival and adaptation of Penicillium chrysogenum to microgravity conditions are not clearly elucidated. A reference genome for Penicillium chrysogenum is not yet available in the NCBI database. Hence, we performed comparative de novo transcriptome analysis of Penicillium chrysogenum grown under microgravity versus normal gravity. In addition, the changes due to microgravity are documented at the molecular level. Increased response to the environmental stimulus, changes in the cell wall component ABC transporter/MFS transporters are noteworthy. Interestingly, sustained increase in the expression of Acyl-coenzyme A: isopenicillin N acyltransferase (Acyltransferase) under microgravity revealed the significance of gravity in the penicillin production which could be exploited industrially.

  10. Comprehensive de novo peptide sequencing from MS/MS pairs generated through complementary collision induced dissociation and 351 nm ultraviolet photodissociation.

    Science.gov (United States)

    Horton, Andrew Pitchford; Robotham, Scott A; Cannon, Joe R; Holden, Dustin D; Marcotte, Edward M; Brodbelt, Jennifer S

    2017-02-24

    We describe a strategy for de novo peptide sequencing based on matched pairs of tandem mass spectra (MS/MS) obtained by collision induced dissociation (CID) and 351 nm ultraviolet photodissociation (UVPD). Each precursor ion is isolated twice with the mass spectrometer switching between CID and UVPD activation modes to obtain a complementary MS/MS pair. To interpret these paired spectra, we modified the UVnovo de novo sequencing software to automatically learn from and interpret fragmentation spectra, provided a representative set of training data. This machine learning procedure, using random forests, synthesizes information from one or multiple complementary spectra, such as the CID/UVPD pairs, into peptide fragmentation site predictions. In doing so, the burden of fragmentation model definition shifts from programmer to machine and opens up the model parameter space for inclusion of nonobvious features and interactions. This spectral synthesis also serves to transform distinct types of spectra into a common representation for subsequent activation-independent processing steps. Then, independent from precursor activation constraints, UVnovo's de novo sequencing procedure generates and scores sequence candidates for each precursor. We demonstrate the combined experimental and computational approach for de novo sequencing using whole cell E. coli lysate. In benchmarks on the CID/UVPD data, UVnovo assigned correct full-length sequences to 83% of the spectral pairs of doubly charged ions with high-confidence database identifications. Considering only top-ranked de novo predictions, 70% of the pairs were deciphered correctly. This de novo sequencing performance exceeds that of PEAKS and PepNovo on the CID spectra and that of UVnovo on CID or UVPD spectra alone. As presented here, the methods for paired CID/UVPD spectral acquisition and interpretation constitute a powerful workflow for high-throughput and accurate de novo peptide sequencing.

  11. De Novo transcriptome sequencing reveals important molecular networks and metabolic pathways of the plant, Chlorophytum borivilianum.

    Science.gov (United States)

    Kalra, Shikha; Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Kumar, Sunil; Kaur, Jagdeep; Ramachandran, Srinivasan; Singh, Kashmir

    2013-01-01

    Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum.

  12. Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using de Novo-Protein Unique Sequence Tags Approach

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yufeng; Tolic, Nikola; Purvine, Samuel O.; Smith, Richard D.

    2010-08-01

    Disulfide bonds are a form of posttranslational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, and specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags) we unambiguously correlated the spectra to specific database proteins. Examination of the UStags’ prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to ~10 kDa.

  13. De novo design of signal sequences to localize cargo to the 1,2-propanediol utilization microcompartment.

    Science.gov (United States)

    Jakobson, Christopher M; Slininger Lee, Marilyn F; Tullman-Ercek, Danielle

    2017-05-01

    Organizing heterologous biosyntheses inside bacterial cells can alleviate common problems owing to toxicity, poor kinetic performance, and cofactor imbalances. A subcellular organelle known as a bacterial microcompartment, such as the 1,2-propanediol utilization microcompartment of Salmonella, is a promising chassis for this strategy. Here we demonstrate de novo design of the N-terminal signal sequences used to direct cargo to these microcompartment organelles. We expand the native repertoire of signal sequences using rational and library-based approaches and show that a canonical leucine-zipper motif can function as a signal sequence for microcompartment localization. Our strategy can be applied to generate new signal sequences localizing arbitrary cargo proteins to the 1,2-propanediol utilization microcompartments. © 2017 The Protein Society.

  14. De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth.

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    Michael A Fisher

    Full Text Available A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >10(6 de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains--ΔserB, ΔgltA, ΔilvA, and Δfes--were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions can be sustained by sequences designed de novo.

  15. Whole-genome sequencing in autism identifies hot spots for de novo germline mutation

    DEFF Research Database (Denmark)

    Michaelson, Jacob J.; Shi, Yujian; Gujral, Madhusudan

    2012-01-01

    De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We...

  16. Image sequence analysis

    CERN Document Server

    1981-01-01

    The processing of image sequences has a broad spectrum of important applica­ tions including target tracking, robot navigation, bandwidth compression of TV conferencing video signals, studying the motion of biological cells using microcinematography, cloud tracking, and highway traffic monitoring. Image sequence processing involves a large amount of data. However, because of the progress in computer, LSI, and VLSI technologies, we have now reached a stage when many useful processing tasks can be done in a reasonable amount of time. As a result, research and development activities in image sequence analysis have recently been growing at a rapid pace. An IEEE Computer Society Workshop on Computer Analysis of Time-Varying Imagery was held in Philadelphia, April 5-6, 1979. A related special issue of the IEEE Transactions on Pattern Anal­ ysis and Machine Intelligence was published in November 1980. The IEEE Com­ puter magazine has also published a special issue on the subject in 1981. The purpose of this book ...

  17. De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

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    Geisa P.C. Evaristo

    2015-09-01

    Full Text Available An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues or CGRPs (37 amino acids and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

  18. High-Quality de Novo Genome Assembly of the Dekkera bruxellensis Yeast Isolate Using Nanopore MinION Sequencing.

    Science.gov (United States)

    Fournier, Téo; Gounot, Jean-Sébastien; Freel, Kelle; Cruaud, Corinne; Lemainque, Arnaud; Aury, Jean-Marc; Wincker, Patrick; Schacherer, Joseph; Friedrich, Anne

    2017-08-09

    Genetic variation in natural populations represents the raw material for phenotypic diversity. Species-wide characterization of genetic variants is crucial to have a deeper insight into the genotype-phenotype relationship. With the advent of new sequencing strategies and more recently the release of long-read sequencing platforms, it is now possible to explore the genetic diversity of any non-model organisms, representing a fundamental resource for biological research. In the frame of population genomic surveys, a first step is to obtain the complete sequence and high quality assembly of a reference genome. Here, we sequenced and assembled a reference genome of the non-conventional Dekkera bruxellensis yeast. While this species is a major cause of wine spoilage, it paradoxically contributes to the specific flavor profile of some Belgium beers. In addition, an extreme karyotype variability is observed across natural isolates, highlighting that D. bruxellensis genome is very dynamic. The whole genome of the D. bruxellensis UMY321 isolate was sequenced using a combination of Nanopore long-read and Illumina short-read sequencing data. We generated the most complete and contiguous de novo assembly of D. bruxellensis to date and obtained a first glimpse into the genomic variability within this species by comparing the sequences of several isolates. This genome sequence is therefore of high value for population genomic surveys and represents a reference to study genome dynamic in this yeast species. Copyright © 2017, G3: Genes, Genomes, Genetics.

  19. De novo clustering methods outperform reference-based methods for assigning 16S rRNA gene sequences to operational taxonomic units

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    Sarah L. Westcott

    2015-12-01

    Full Text Available Background. 16S rRNA gene sequences are routinely assigned to operational taxonomic units (OTUs that are then used to analyze complex microbial communities. A number of methods have been employed to carry out the assignment of 16S rRNA gene sequences to OTUs leading to confusion over which method is optimal. A recent study suggested that a clustering method should be selected based on its ability to generate stable OTU assignments that do not change as additional sequences are added to the dataset. In contrast, we contend that the quality of the OTU assignments, the ability of the method to properly represent the distances between the sequences, is more important.Methods. Our analysis implemented six de novo clustering algorithms including the single linkage, complete linkage, average linkage, abundance-based greedy clustering, distance-based greedy clustering, and Swarm and the open and closed-reference methods. Using two previously published datasets we used the Matthew’s Correlation Coefficient (MCC to assess the stability and quality of OTU assignments.Results. The stability of OTU assignments did not reflect the quality of the assignments. Depending on the dataset being analyzed, the average linkage and the distance and abundance-based greedy clustering methods generated OTUs that were more likely to represent the actual distances between sequences than the open and closed-reference methods. We also demonstrated that for the greedy algorithms VSEARCH produced assignments that were comparable to those produced by USEARCH making VSEARCH a viable free and open source alternative to USEARCH. Further interrogation of the reference-based methods indicated that when USEARCH or VSEARCH were used to identify the closest reference, the OTU assignments were sensitive to the order of the reference sequences because the reference sequences can be identical over the region being considered. More troubling was the observation that while both USEARCH and

  20. Whole genome sequencing reveals a de novo SHANK3 mutation in familial autism spectrum disorder.

    Directory of Open Access Journals (Sweden)

    Sergio I Nemirovsky

    Full Text Available Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD. Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS for the diagnostic approach to ASD.We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents.Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6.We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder.

  1. De Novo Transcriptome Analysis and Detection of Antimicrobial Peptides of the American Cockroach Periplaneta americana (Linnaeus.

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    In-Woo Kim

    Full Text Available Cockroaches are surrogate hosts for microbes that cause many human diseases. In spite of their generally destructive nature, cockroaches have recently been found to harbor potentially beneficial and medically useful substances such as drugs and allergens. However, genomic information for the American cockroach (Periplaneta americana is currently unavailable; therefore, transcriptome and gene expression profiling is needed as an important resource to better understand the fundamental biological mechanisms of this species, which would be particularly useful for the selection of novel antimicrobial peptides. Thus, we performed de novo transcriptome analysis of P. americana that were or were not immunized with Escherichia coli. Using an Illumina HiSeq sequencer, we generated a total of 9.5 Gb of sequences, which were assembled into 85,984 contigs and functionally annotated using Basic Local Alignment Search Tool (BLAST, Gene Ontology (GO, and Kyoto Encyclopedia of Genes and Genomes (KEGG database terms. Finally, using an in silico antimicrobial peptide prediction method, 86 antimicrobial peptide candidates were predicted from the transcriptome, and 21 of these peptides were experimentally validated for their antimicrobial activity against yeast and gram positive and -negative bacteria by a radial diffusion assay. Notably, 11 peptides showed strong antimicrobial activities against these organisms and displayed little or no cytotoxic effects in the hemolysis and cell viability assay. This work provides prerequisite baseline data for the identification and development of novel antimicrobial peptides, which is expected to provide a better understanding of the phenomenon of innate immunity in similar species.

  2. Simultaneous transcriptome analysis of Sorghum and Bipolaris sorghicola by using RNA-seq in combination with de novo transcriptome assembly.

    Directory of Open Access Journals (Sweden)

    Takayuki Yazawa

    Full Text Available The recent development of RNA sequencing (RNA-seq technology has enabled us to analyze the transcriptomes of plants and their pathogens simultaneously. However, RNA-seq often relies on aligning reads to a reference genome and is thus unsuitable for analyzing most plant pathogens, as their genomes have not been fully sequenced. Here, we analyzed the transcriptomes of Sorghum bicolor (L. Moench and its pathogen Bipolaris sorghicola simultaneously by using RNA-seq in combination with de novo transcriptome assembly. We sequenced the mixed transcriptome of the disease-resistant sorghum cultivar SIL-05 and B. sorghicola in infected leaves in the early stages of infection (12 and 24 h post-inoculation by using Illumina mRNA-Seq technology. Sorghum gene expression was quantified by aligning reads to the sorghum reference genome. For B. sorghicola, reads that could not be aligned to the sorghum reference genome were subjected to de novo transcriptome assembly. We identified genes of B. sorghicola for growth of this fungus in sorghum, as well as genes in sorghum for the defense response. The genes of B. sorghicola included those encoding Woronin body major protein, LysM domain-containing intracellular hyphae protein, transcriptional factors CpcA and HacA, and plant cell-wall degrading enzymes. The sorghum genes included those encoding two receptors of the simple eLRR domain protein family, transcription factors that are putative orthologs of OsWRKY45 and OsWRKY28 in rice, and a class III peroxidase that is a homolog involved in disease resistance in the Poaceae. These defense-related genes were particularly strongly induced among paralogs annotated in the sorghum genome. Thus, in the absence of genome sequences for the pathogen, simultaneous transcriptome analysis of plant and pathogen by using de novo assembly was useful for identifying putative key genes in the plant-pathogen interaction.

  3. A Proteomic Workflow Using High-Throughput De Novo Sequencing Towards Complementation of Genome Information for Improved Comparative Crop Science.

    Science.gov (United States)

    Turetschek, Reinhard; Lyon, David; Desalegn, Getinet; Kaul, Hans-Peter; Wienkoop, Stefanie

    2016-01-01

    The proteomic study of non-model organisms, such as many crop plants, is challenging due to the lack of comprehensive genome information. Changing environmental conditions require the study and selection of adapted cultivars. Mutations, inherent to cultivars, hamper protein identification and thus considerably complicate the qualitative and quantitative comparison in large-scale systems biology approaches. With this workflow, cultivar-specific mutations are detected from high-throughput comparative MS analyses, by extracting sequence polymorphisms with de novo sequencing. Stringent criteria are suggested to filter for confidential mutations. Subsequently, these polymorphisms complement the initially used database, which is ready to use with any preferred database search algorithm. In our example, we thereby identified 26 specific mutations in two cultivars of Pisum sativum and achieved an increased number (17 %) of peptide spectrum matches.

  4. RNA-seq analysis and de novo transcriptome assembly of Jerusalem artichoke (Helianthus tuberosus Linne).

    Science.gov (United States)

    Jung, Won Yong; Lee, Sang Sook; Kim, Chul Wook; Kim, Hyun-Soon; Min, Sung Ran; Moon, Jae Sun; Kwon, Suk-Yoon; Jeon, Jae-Heung; Cho, Hye Sun

    2014-01-01

    Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

  5. RNA-seq analysis and de novo transcriptome assembly of Jerusalem artichoke (Helianthus tuberosus Linne.

    Directory of Open Access Journals (Sweden)

    Won Yong Jung

    Full Text Available Jerusalem artichoke (Helianthus tuberosus L. has long been cultivated as a vegetable and as a source of fructans (inulin for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development. Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke.

  6. Direct chloroplast sequencing: comparison of sequencing platforms and analysis tools for whole chloroplast barcoding.

    Directory of Open Access Journals (Sweden)

    Marta Brozynska

    Full Text Available Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina and Ion Torrent (Life Technology sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare. Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.

  7. Exome sequencing identifies de novo gain of function missense mutation in KCND2 in identical twins with autism and seizures that slows potassium channel inactivation.

    Science.gov (United States)

    Lee, Hane; Lin, Meng-chin A; Kornblum, Harley I; Papazian, Diane M; Nelson, Stanley F

    2014-07-01

    Numerous studies and case reports show comorbidity of autism and epilepsy, suggesting some common molecular underpinnings of the two phenotypes. However, the relationship between the two, on the molecular level, remains unclear. Here, whole exome sequencing was performed on a family with identical twins affected with autism and severe, intractable seizures. A de novo variant was identified in the KCND2 gene, which encodes the Kv4.2 potassium channel. Kv4.2 is a major pore-forming subunit in somatodendritic subthreshold A-type potassium current (ISA) channels. The de novo mutation p.Val404Met is novel and occurs at a highly conserved residue within the C-terminal end of the transmembrane helix S6 region of the ion permeation pathway. Functional analysis revealed the likely pathogenicity of the variant in that the p.Val404Met mutant construct showed significantly slowed inactivation, either by itself or after equimolar coexpression with the wild-type Kv4.2 channel construct consistent with a dominant effect. Further, the effect of the mutation on closed-state inactivation was evident in the presence of auxiliary subunits that associate with Kv4 subunits to form ISA channels in vivo. Discovery of a functionally relevant novel de novo variant, coupled with physiological evidence that the mutant protein disrupts potassium current inactivation, strongly supports KCND2 as the causal gene for epilepsy in this family. Interaction of KCND2 with other genes implicated in autism and the role of KCND2 in synaptic plasticity provide suggestive evidence of an etiological role in autism.

  8. Complete genome sequence of novel carbon monoxide oxidizing bacteria Citrobacter amalonaticus Y19, assembled de novo.

    Science.gov (United States)

    Ainala, Satish Kumar; Seol, Eunhee; Park, Sunghoon

    2015-10-10

    We report here the complete genome sequence of Citrobacter amalonaticus Y19 isolated from an anaerobic digester. PacBio single-molecule real-time (SMRT) sequencing was employed, resulting in a single scaffold of 5.58Mb. The sequence of a mega plasmid of 291Kb size is also presented.

  9. De novo assembly and transcriptome analysis of sclerotial development in Wolfiporia cocos.

    Science.gov (United States)

    Wu, Yayun; Zhu, Wenjun; Wei, Wei; Zhao, Xiaolong; Wang, Qi; Zeng, Wanyong; Zheng, Yonglian; Chen, Ping; Zhang, Shaopeng

    2016-08-22

    Wolfiporia cocos Ryvarden et Gilbertson, a well-known medicinal fungus in the Basidiomycetes, is widely distributed in East Asia. Its dried sclerotium, which is known as Fuling in China, has been used as a traditional crude drug in Chinese traditional medicine for thousand years. However, little is known about how the sclerotium is developed at the genetic level. In this study, the de novo sequencing of sclerotia of W. cocos (S1_initial stage; S2_developmental stage and S3_mature stage) was carried out by illumina HiSeq 2000 technology. 27,438 unigenes were assembled from ~30Gbp raw data, and 12,093 unigenes were significantly annotated. The analysis of expression profiles during development returned 304 differentially expressed genes (DEGs), which were clustered into four different groups according to their expression trends. Especially for the maturation stage (S3), the sclerotium exhibited a markedly different expression profile from other stages. We further showed that peroxisome, unsaturation of fatty acids and degradation pathway were respectively prevalent in S1, S2 and S3 stages as evidenced by enrichment analysis. To our knowledge, this study represents the first report of sclerotial development transcriptomics in W. cocos. The obtained results provide novel insights into the developmental biology of the sclerotia, which is helpful for future studies about cultivation and breeding of W. cocos.

  10. Whole exome sequencing reveals a novel de novo FOXC1 mutation in a patient with unrecognized Axenfeld-Rieger syndrome and glaucoma.

    Science.gov (United States)

    Pasutto, F; Mauri, L; Popp, B; Sticht, H; Ekici, A; Piozzi, E; Bonfante, A; Penco, S; Schlötzer-Schrehardt, U; Reis, A

    2015-08-15

    We report the identification of a novel mutation in the fork-head box C1 (FOXC1) gene which occurred de novo in an Italian patient with unrecognized Axenfeld-Rieger syndrome. He was previously diagnosed as having late recognized primary congenital glaucoma at the age of 14 years and was subsequently subjected to multiple surgical interventions due to uncontrolled intraocular pressure and progressive visual field loss. After exclusion of mutations in CYP1B1 and MYOC, trio-whole-exome sequencing revealed de novo in frame deletion in the coding region of the FOXC1 gene (c.407_409delGTC, p.V137del) leading to a deletion of the evolutionary conserved amino acid Valine at position 137 of the protein. Molecular modeling predicted that Val137 deletion impairs FOXC1 DNA-binding capacity and transcriptional activation. Since loss-of-function mutations in FOXC1 are associated with Axenfeld-Rieger syndrome, the genetic findings in combination with re-evaluation of the patient's clinical data resulted in a corrected diagnosis of Axenfeld-Rieger syndrome with developmental glaucoma. We therefore suggest that in addition to CYP1B1 and MYOC, FOXC1 should be included in the genetic analysis of cases with unclear glaucomatous phenotypes to ensure proper diagnosis, adequate treatment and appropriate genetic counseling.

  11. Terminal sequence importance of de novo proteins from binary-patterned library: stable artificial proteins with 11- or 12-amino acid alphabet.

    Science.gov (United States)

    Okura, Hiromichi; Takahashi, Tsuyoshi; Mihara, Hisakazu

    2012-06-01

    Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

  12. De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.

    Directory of Open Access Journals (Sweden)

    Li Dejun

    2012-05-01

    Full Text Available Abstract Background In rubber tree, bark is one of important agricultural and biological organs. However, the molecular mechanism involved in the bark formation and development in rubber tree remains largely unknown, which is at least partially due to lack of bark transcriptomic and genomic information. Therefore, it is necessary to carried out high-throughput transcriptome sequencing of rubber tree bark to generate enormous transcript sequences for the functional characterization and molecular marker development. Results In this study, more than 30 million sequencing reads were generated using Illumina paired-end sequencing technology. In total, 22,756 unigenes with an average length of 485 bp were obtained with de novo assembly. The similarity search indicated that 16,520 and 12,558 unigenes showed significant similarities to known proteins from NCBI non-redundant and Swissprot protein databases, respectively. Among these annotated unigenes, 6,867 and 5,559 unigenes were separately assigned to Gene Ontology (GO and Clusters of Orthologous Group (COG. When 22,756 unigenes searched against the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG database, 12,097 unigenes were assigned to 5 main categories including 123 KEGG pathways. Among the main KEGG categories, metabolism was the biggest category (9,043, 74.75%, suggesting the active metabolic processes in rubber tree bark. In addition, a total of 39,257 EST-SSRs were identified from 22,756 unigenes, and the characterizations of EST-SSRs were further analyzed in rubber tree. 110 potential marker sites were randomly selected to validate the assembly quality and develop EST-SSR markers. Among 13 Hevea germplasms, PCR success rate and polymorphism rate of 110 markers were separately 96.36% and 55.45% in this study. Conclusion By assembling and analyzing de novo transcriptome sequencing data, we reported the comprehensive functional characterization of rubber tree bark. This research

  13. De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis.

    Science.gov (United States)

    Hyun, Tae Kyung; Rim, Yeonggil; Jang, Hui-Jeong; Kim, Cheol Hong; Park, Jongsun; Kumar, Ritesh; Lee, Sunghoon; Kim, Byung Chul; Bhak, Jong; Nguyen-Quoc, Binh; Kim, Seon-Won; Lee, Sang Yeol; Kim, Jae-Yean

    2012-07-01

    The ripe fruit of Momordica cochinchinensis Spreng, known as gac, is featured by very high carotenoid content. Although this plant might be a good resource for carotenoid metabolic engineering, so far, the genes involved in the carotenoid metabolic pathways in gac were unidentified due to lack of genomic information in the public database. In order to expedite the process of gene discovery, we have undertaken Illumina deep sequencing of mRNA prepared from aril of gac fruit. From 51,446,670 high-quality reads, we obtained 81,404 assembled unigenes with average length of 388 base pairs. At the protein level, gac aril transcripts showed about 81.5% similarity with cucumber proteomes. In addition 17,104 unigenes have been assigned to specific metabolic pathways in Kyoto Encyclopedia of Genes and Genomes, and all of known enzymes involved in terpenoid backbones biosynthetic and carotenoid biosynthetic pathways were also identified in our library. To analyze the relationship between putative carotenoid biosynthesis genes and alteration of carotenoid content during fruit ripening, digital gene expression analysis was performed on three different ripening stages of aril. This study has revealed putative phytoene synthase, 15-cis-phytone desaturase, zeta-carotene desaturase, carotenoid isomerase and lycopene epsilon cyclase might be key factors for controlling carotenoid contents during aril ripening. Taken together, this study has also made availability of a large gene database. This unique information for gac gene discovery would be helpful to facilitate functional studies for improving carotenoid quantities.

  14. Microsatellite loci and the complete mitochondrial DNA sequence characterized through next generation sequencing and de novo genome assembly for the critically endangered orange-bellied parrot, Neophema chrysogaster.

    Science.gov (United States)

    Miller, Adam D; Good, Robert T; Coleman, Rhys A; Lancaster, Melanie L; Weeks, Andrew R

    2013-01-01

    A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.

  15. Sequencing, De Novo assembly and annotation of the Colorado Potato Beetle, Leptinotarsa decemlineata, Transcriptome.

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    Abhishek Kumar

    Full Text Available BACKGROUND: The Colorado potato beetle (Leptinotarsa decemlineata is a major pest and a serious threat to potato cultivation throughout the northern hemisphere. Despite its high importance for invasion biology, phenology and pest management, little is known about L. decemlineata from a genomic perspective. We subjected European L. decemlineata adult and larval transcriptome samples to 454-FLX massively-parallel DNA sequencing to characterize a basal set of genes from this species. We created a combined assembly of the adult and larval datasets including the publicly available midgut larval Roche 454 reads and provided basic annotation. We were particularly interested in diapause-specific genes and genes involved in pesticide and Bacillus thuringiensis (Bt resistance. RESULTS: Using 454-FLX pyrosequencing, we obtained a total of 898,048 reads which, together with the publicly available 804,056 midgut larval reads, were assembled into 121,912 contigs. We established a repository of genes of interest, with 101 out of the 108 diapause-specific genes described in Drosophila montana; and 621 contigs involved in insecticide resistance, including 221 CYP450, 45 GSTs, 13 catalases, 15 superoxide dismutases, 22 glutathione peroxidases, 194 esterases, 3 ADAM metalloproteases, 10 cadherins and 98 calmodulins. We found 460 putative miRNAs and we predicted a significant number of single nucleotide polymorphisms (29,205 and microsatellite loci (17,284. CONCLUSIONS: This report of the assembly and annotation of the transcriptome of L. decemlineata offers new insights into diapause-associated and insecticide-resistance-associated genes in this species and provides a foundation for comparative studies with other species of insects. The data will also open new avenues for researchers using L. decemlineata as a model species, and for pest management research. Our results provide the basis for performing future gene expression and functional analysis in L

  16. Sequencing, De Novo Assembly, and Annotation of the Transcriptome of the Endangered Freshwater Pearl Bivalve, Cristaria plicata, Provides Novel Insights into Functional Genes and Marker Discovery.

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    Bharat Bhusan Patnaik

    Full Text Available The freshwater mussel Cristaria plicata (Bivalvia: Eulamellibranchia: Unionidae, is an economically important species in molluscan aquaculture due to its use in pearl farming. The species have been listed as endangered in South Korea due to the loss of natural habitats caused by anthropogenic activities. The decreasing population and a lack of genomic information on the species is concerning for environmentalists and conservationists. In this study, we conducted a de novo transcriptome sequencing and annotation analysis of C. plicata using Illumina HiSeq 2500 next-generation sequencing (NGS technology, the Trinity assembler, and bioinformatics databases to prepare a sustainable resource for the identification of candidate genes involved in immunity, defense, and reproduction.The C. plicata transcriptome analysis included a total of 286,152,584 raw reads and 281,322,837 clean reads. The de novo assembly identified a total of 453,931 contigs and 374,794 non-redundant unigenes with average lengths of 731.2 and 737.1 bp, respectively. Furthermore, 100% coverage of C. plicata mitochondrial genes within two unigenes supported the quality of the assembler. In total, 84,274 unigenes showed homology to entries in at least one database, and 23,246 unigenes were allocated to one or more Gene Ontology (GO terms. The most prominent GO biological process, cellular component, and molecular function categories (level 2 were cellular process, membrane, and binding, respectively. A total of 4,776 unigenes were mapped to 123 biological pathways in the KEGG database. Based on the GO terms and KEGG annotation, the unigenes were suggested to be involved in immunity, stress responses, sex-determination, and reproduction. A total of 17,251 cDNA simple sequence repeats (cSSRs were identified from 61,141 unigenes (size of >1 kb with the most abundant being dinucleotide repeats.This dataset represents the first transcriptome analysis of the endangered mollusc, C. plicata

  17. De Novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon's blood.

    Science.gov (United States)

    Zhu, Jia-Hong; Cao, Tian-Jun; Dai, Hao-Fu; Li, Hui-Liang; Guo, Dong; Mei, Wen-Li; Peng, Shi-Qing

    2016-12-06

    Dragon's blood is a red resin mainly extracted from Dracaena plants, and has been widely used as a traditional medicine in East and Southeast Asia. The major components of dragon's blood are flavonoids. Owing to a lack of Dracaena plants genomic information, the flavonoids biosynthesis and regulation in Dracaena plants remain unknown. In this study, three cDNA libraries were constructed from the stems of D. cambodiana after injecting the inducer. Approximately 266.57 million raw sequencing reads were de novo assembled into 198,204 unigenes, of which 34,873 unique sequences were annotated in public protein databases. Many candidate genes involved in flavonoid accumulation were identified. Differential expression analysis identified 20 genes involved in flavonoid biosynthesis, 27 unigenes involved in flavonoid modification and 68 genes involved in flavonoid transport that were up-regulated in the stems of D. cambodiana after injecting the inducer, consistent with the accumulation of flavonoids. Furthermore, we have revealed the differential expression of transcripts encoding for transcription factors (MYB, bHLH and WD40) involved in flavonoid metabolism. These de novo transcriptome data sets provide insights on pathways and molecular regulation of flavonoid biosynthesis and transport, and improve our understanding of molecular mechanisms of dragon's blood formation in D. cambodiana.

  18. SEQUENCING AND DE NOVO DRAFT ASSEMBLIES OF A FATHEAD MINNOW (Pimpehales promelas) reference genome

    Data.gov (United States)

    U.S. Environmental Protection Agency — The dataset provides the URLs for accessing the genome sequence data and two draft assemblies as well as fathead minnow genotyping data associated with estimating...

  19. Rare variant detection using family-based sequencing analysis.

    Science.gov (United States)

    Peng, Gang; Fan, Yu; Palculict, Timothy B; Shen, Peidong; Ruteshouser, E Cristy; Chi, Aung-Kyaw; Davis, Ronald W; Huff, Vicki; Scharfe, Curt; Wang, Wenyi

    2013-03-05

    Next-generation sequencing is revolutionizing genomic analysis, but this analysis can be compromised by high rates of missing true variants. To develop a robust statistical method capable of identifying variants that would otherwise not be called, we conducted sequence data simulations and both whole-genome and targeted sequencing data analysis of 28 families. Our method (Family-Based Sequencing Program, FamSeq) integrates Mendelian transmission information and raw sequencing reads. Sequence analysis using FamSeq reduced the number of false negative variants by 14-33% as assessed by HapMap sample genotype confirmation. In a large family affected with Wilms tumor, 84% of variants uniquely identified by FamSeq were confirmed by Sanger sequencing. In children with early-onset neurodevelopmental disorders from 26 families, de novo variant calls in disease candidate genes were corrected by FamSeq as mendelian variants, and the number of uniquely identified variants in affected individuals increased proportionally as additional family members were included in the analysis. To gain insight into maximizing variant detection, we studied factors impacting actual improvements of family-based calling, including pedigree structure, allele frequency (common vs. rare variants), prior settings of minor allele frequency, sequence signal-to-noise ratio, and coverage depth (∼20× to >200×). These data will help guide the design, analysis, and interpretation of family-based sequencing studies to improve the ability to identify new disease-associated genes.

  20. Transcriptome Profile of the Asian Giant Hornet (Vespa mandarinia) Using Illumina HiSeq 4000 Sequencing: De Novo Assembly, Functional Annotation, and Discovery of SSR Markers.

    Science.gov (United States)

    Patnaik, Bharat Bhusan; Park, So Young; Kang, Se Won; Hwang, Hee-Ju; Wang, Tae Hun; Park, Eun Bi; Chung, Jong Min; Song, Dae Kwon; Kim, Changmu; Kim, Soonok; Lee, Jae Bong; Jeong, Heon Cheon; Park, Hong Seog; Han, Yeon Soo; Lee, Yong Seok

    2016-01-01

    Vespa mandarinia found in the forests of East Asia, including Korea, occupies the highest rank in the arthropod food web within its geographical range. It serves as a source of nutrition in the form of Vespa amino acid mixture and is listed as a threatened species, although no conservation measures have been implemented. Here, we performed de novo assembly of the V. mandarinia transcriptome by Illumina HiSeq 4000 sequencing. Over 60 million raw reads and 59,184,811 clean reads were obtained. After assembly, a total of 66,837 unigenes were clustered, 40,887, 44,455, and 22,390 of which showed homologous matches against the PANM, Unigene, and KOG databases, respectively. A total of 15,675 unigenes were assigned to Gene Ontology terms, and 5,132 unigenes were mapped to 115 KEGG pathways. The zinc finger domain (C2H2-like), serine/threonine/dual specificity protein kinase domain, and RNA recognition motif domain were among the top InterProScan domains predicted for V. mandarinia sequences. Among the unigenes, we identified 534,922 cDNA simple sequence repeats as potential markers. This is the first transcriptomic analysis of the wasp V. mandarinia using Illumina HiSeq 4000. The obtained datasets should promote the search for new genes to understand the physiological attributes of this wasp.

  1. Transcriptome Profile of the Asian Giant Hornet (Vespa mandarinia Using Illumina HiSeq 4000 Sequencing: De Novo Assembly, Functional Annotation, and Discovery of SSR Markers

    Directory of Open Access Journals (Sweden)

    Bharat Bhusan Patnaik

    2016-01-01

    Full Text Available Vespa mandarinia found in the forests of East Asia, including Korea, occupies the highest rank in the arthropod food web within its geographical range. It serves as a source of nutrition in the form of Vespa amino acid mixture and is listed as a threatened species, although no conservation measures have been implemented. Here, we performed de novo assembly of the V. mandarinia transcriptome by Illumina HiSeq 4000 sequencing. Over 60 million raw reads and 59,184,811 clean reads were obtained. After assembly, a total of 66,837 unigenes were clustered, 40,887, 44,455, and 22,390 of which showed homologous matches against the PANM, Unigene, and KOG databases, respectively. A total of 15,675 unigenes were assigned to Gene Ontology terms, and 5,132 unigenes were mapped to 115 KEGG pathways. The zinc finger domain (C2H2-like, serine/threonine/dual specificity protein kinase domain, and RNA recognition motif domain were among the top InterProScan domains predicted for V. mandarinia sequences. Among the unigenes, we identified 534,922 cDNA simple sequence repeats as potential markers. This is the first transcriptomic analysis of the wasp V. mandarinia using Illumina HiSeq 4000. The obtained datasets should promote the search for new genes to understand the physiological attributes of this wasp.

  2. Discovery of genes related to insecticide resistance in Bactrocera dorsalis by functional genomic analysis of a de novo assembled transcriptome.

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    Ju-Chun Hsu

    Full Text Available Insecticide resistance has recently become a critical concern for control of many insect pest species. Genome sequencing and global quantization of gene expression through analysis of the transcriptome can provide useful information relevant to this challenging problem. The oriental fruit fly, Bactrocera dorsalis, is one of the world's most destructive agricultural pests, and recently it has been used as a target for studies of genetic mechanisms related to insecticide resistance. However, prior to this study, the molecular data available for this species was largely limited to genes identified through homology. To provide a broader pool of gene sequences of potential interest with regard to insecticide resistance, this study uses whole transcriptome analysis developed through de novo assembly of short reads generated by next-generation sequencing (NGS. The transcriptome of B. dorsalis was initially constructed using Illumina's Solexa sequencing technology. Qualified reads were assembled into contigs and potential splicing variants (isotigs. A total of 29,067 isotigs have putative homologues in the non-redundant (nr protein database from NCBI, and 11,073 of these correspond to distinct D. melanogaster proteins in the RefSeq database. Approximately 5,546 isotigs contain coding sequences that are at least 80% complete and appear to represent B. dorsalis genes. We observed a strong correlation between the completeness of the assembled sequences and the expression intensity of the transcripts. The assembled sequences were also used to identify large numbers of genes potentially belonging to families related to insecticide resistance. A total of 90 P450-, 42 GST-and 37 COE-related genes, representing three major enzyme families involved in insecticide metabolism and resistance, were identified. In addition, 36 isotigs were discovered to contain target site sequences related to four classes of resistance genes. Identified sequence motifs were also

  3. Discovery of Genes Related to Insecticide Resistance in Bactrocera dorsalis by Functional Genomic Analysis of a De Novo Assembled Transcriptome

    Science.gov (United States)

    Hsu, Ju-Chun; Wu, Wen-Jer; Feng, Hai-Tung; Haymer, David S.; Chen, Chien-Yu

    2012-01-01

    Insecticide resistance has recently become a critical concern for control of many insect pest species. Genome sequencing and global quantization of gene expression through analysis of the transcriptome can provide useful information relevant to this challenging problem. The oriental fruit fly, Bactrocera dorsalis, is one of the world's most destructive agricultural pests, and recently it has been used as a target for studies of genetic mechanisms related to insecticide resistance. However, prior to this study, the molecular data available for this species was largely limited to genes identified through homology. To provide a broader pool of gene sequences of potential interest with regard to insecticide resistance, this study uses whole transcriptome analysis developed through de novo assembly of short reads generated by next-generation sequencing (NGS). The transcriptome of B. dorsalis was initially constructed using Illumina's Solexa sequencing technology. Qualified reads were assembled into contigs and potential splicing variants (isotigs). A total of 29,067 isotigs have putative homologues in the non-redundant (nr) protein database from NCBI, and 11,073 of these correspond to distinct D. melanogaster proteins in the RefSeq database. Approximately 5,546 isotigs contain coding sequences that are at least 80% complete and appear to represent B. dorsalis genes. We observed a strong correlation between the completeness of the assembled sequences and the expression intensity of the transcripts. The assembled sequences were also used to identify large numbers of genes potentially belonging to families related to insecticide resistance. A total of 90 P450-, 42 GST-and 37 COE-related genes, representing three major enzyme families involved in insecticide metabolism and resistance, were identified. In addition, 36 isotigs were discovered to contain target site sequences related to four classes of resistance genes. Identified sequence motifs were also analyzed to

  4. Sequencing and de novo draft assemblies of a fathead minnow (Pimephales promelas) reference genome.

    Science.gov (United States)

    Burns, Frank R; Cogburn, Amarin L; Ankley, Gerald T; Villeneuve, Daniel L; Waits, Eric; Chang, Yun-Juan; Llaca, Victor; Deschamps, Stephane D; Jackson, Raymond E; Hoke, Robert Alan

    2016-01-01

    The present study was undertaken to provide the foundation for development of genome-scale resources for the fathead minnow (Pimephales promelas), an important model organism widely used in both aquatic toxicology research and regulatory testing. The authors report on the first sequencing and 2 draft assemblies for the reference genome of this species. Approximately 120× sequence coverage was achieved via Illumina sequencing of a combination of paired-end, mate-pair, and fosmid libraries. Evaluation and comparison of these assemblies demonstrate that they are of sufficient quality to be useful for genome-enabled studies, with 418 of 458 (91%) conserved eukaryotic genes mapping to at least 1 of the assemblies. In addition to its immediate utility, the present work provides a strong foundation on which to build further refinements of a reference genome for the fathead minnow.

  5. Genomic resources for water yam (Dioscorea alata L.): analyses of EST-Sequences, De Novo sequencing and GBS libraries

    Science.gov (United States)

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources such as SSRs, SNPs and InDels in several model and non-model plant species. Yam (Dioscorea spp.) i...

  6. De novo assembly, gene annotation, and marker discovery in stored-product pest Liposcelis entomophila (Enderlein using transcriptome sequences.

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    Dan-Dan Wei

    Full Text Available BACKGROUND: As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels. METHODOLOGY/PRINCIPAL FINDINGS: We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61% unigenes were matched to known proteins in the NCBI non-redundant (Nr protein database. These unigenes were further functionally annotated with gene ontology (GO, cluster of orthologous groups of proteins (COG, and Kyoto Encyclopedia of Genes and Genomes (KEGG databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST genes, 19 putative carboxyl/cholinesterase (CCE genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future. CONCLUSIONS/SIGNIFICANCE: We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying

  7. Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity.

    Science.gov (United States)

    Astafieva, Alexandra A; Enyenihi, Atim A; Rogozhin, Eugene A; Kozlov, Sergey A; Grishin, Eugene V; Odintsova, Tatyana I; Zubarev, Roman A; Egorov, Tsezi A

    2015-09-01

    Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. De Novo Assembly of Bitter Gourd Transcriptomes: Gene Expression and Sequence Variations in Gynoecious and Monoecious Lines.

    Science.gov (United States)

    Shukla, Anjali; Singh, V K; Bharadwaj, D R; Kumar, Rajesh; Rai, Ashutosh; Rai, A K; Mugasimangalam, Raja; Parameswaran, Sriram; Singh, Major; Naik, P S

    2015-01-01

    Bitter gourd (Momordica charantia L.) is a nutritious vegetable crop of Asian origin, used as a medicinal herb in Indian and Chinese traditional medicine. Molecular breeding in bitter gourd is in its infancy, due to limited molecular resources, particularly on functional markers for traits such as gynoecy. We performed de novo transcriptome sequencing of bitter gourd using Illumina next-generation sequencer, from root, flower buds, stem and leaf samples of gynoecious line (Gy323) and a monoecious line (DRAR1). A total of 65,540 transcripts for Gy323 and 61,490 for DRAR1 were obtained. Comparisons revealed SNP and SSR variations between these lines and, identification of gene classes. Based on available transcripts we identified 80 WRKY transcription factors, several reported in responses to biotic and abiotic stresses; 56 ARF genes which play a pivotal role in auxin-regulated gene expression and development. The data presented will be useful in both functions studies and breeding programs in bitter gourd.

  9. Sequencing and De novo Draft Assemblies of the Fathead Minnow (Pimphales promelas)Reference Genome

    Science.gov (United States)

    This study was undertaken to develop genome-scale resources for the fathead minnow (Pimphales promelas) an important model organism widely used in both aquatic ecotoxicology research and in regulatory toxicity testing. We report on the first sequencing and two draft assemblies fo...

  10. Sequencing and De novo Draft Assemblies of the Fathead Minnow (Pimphales promelas)Reference Genome

    Science.gov (United States)

    This study was undertaken to develop genome-scale resources for the fathead minnow (Pimphales promelas) an important model organism widely used in both aquatic ecotoxicology research and in regulatory toxicity testing. We report on the first sequencing and two draft assemblies fo...

  11. Sequencing and de novo assembly of 150 genomes from Denmark as a population reference

    DEFF Research Database (Denmark)

    Maretty, Lasse; Jensen, Jacob Malte; Petersen, Bent

    2017-01-01

    Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome ...

  12. RNA-seq analysis of Quercus pubescens Leaves: de novo transcriptome assembly, annotation and functional markers development.

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    Sara Torre

    Full Text Available Quercus pubescens Willd., a species distributed from Spain to southwest Asia, ranks high for drought tolerance among European oaks. Q. pubescens performs a role of outstanding significance in most Mediterranean forest ecosystems, but few mechanistic studies have been conducted to explore its response to environmental constrains, due to the lack of genomic resources. In our study, we performed a deep transcriptomic sequencing in Q. pubescens leaves, including de novo assembly, functional annotation and the identification of new molecular markers. Our results are a pre-requisite for undertaking molecular functional studies, and may give support in population and association genetic studies. 254,265,700 clean reads were generated by the Illumina HiSeq 2000 platform, with an average length of 98 bp. De novo assembly, using CLC Genomics, produced 96,006 contigs, having a mean length of 618 bp. Sequence similarity analyses against seven public databases (Uniprot, NR, RefSeq and KOGs at NCBI, Pfam, InterPro and KEGG resulted in 83,065 transcripts annotated with gene descriptions, conserved protein domains, or gene ontology terms. These annotations and local BLAST allowed identify genes specifically associated with mechanisms of drought avoidance. Finally, 14,202 microsatellite markers and 18,425 single nucleotide polymorphisms (SNPs were, in silico, discovered in assembled and annotated sequences. We completed a successful global analysis of the Q. pubescens leaf transcriptome using RNA-seq. The assembled and annotated sequences together with newly discovered molecular markers provide genomic information for functional genomic studies in Q. pubescens, with special emphasis to response mechanisms to severe constrain of the Mediterranean climate. Our tools enable comparative genomics studies on other Quercus species taking advantage of large intra-specific ecophysiological differences.

  13. Molecular analysis of "de novo" purine biosynthesis in solanaceous species and in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    van der Graaff, Eric; Hooykaas, Paul; Lein, Wolfgang

    2004-01-01

    , microorganisms and Arabidopsis, the first plant species with a completely sequenced genome, shows that plants principally use the same biochemical steps to synthesize purine nucleotides and possess all the essential genes and enzymes. Here we report on the cloning and molecular analysis of the complete purine...... biosynthesis pathway in plants, and the in planta functional analysis of PRPP (5-phosphoribosyl-1-pyrophoshate) amidotransferase (ATase), catalyzing the first committed step of the "de novo" purine biosynthesis. The cloning of the genes involved in the purine biosynthesis pathway was attained by a screening...... strategy with heterologous cDNA probes and by using S. cerevisiae mutants for complementation. Southern hybridization showed a complex genomic organization for these genes in solanaceous species and their organ- and developmental specific expression was analyzed by Northern hybridization. The specific role...

  14. De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody.

    Science.gov (United States)

    Bogdanoff, Walter A; Morgenstern, David; Bern, Marshall; Ueberheide, Beatrix M; Sanchez-Fauquier, Alicia; DuBois, Rebecca M

    2016-05-13

    Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent.

  15. De novo genome assembly of the economically important weed horseweed using integrated data from multiple sequencing platforms.

    Science.gov (United States)

    Peng, Yanhui; Lai, Zhao; Lane, Thomas; Nageswara-Rao, Madhugiri; Okada, Miki; Jasieniuk, Marie; O'Geen, Henriette; Kim, Ryan W; Sammons, R Douglas; Rieseberg, Loren H; Stewart, C Neal

    2014-11-01

    Horseweed (Conyza canadensis), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n = 2x = 18), with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic bases of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000, and PacBio RS) using various libraries with different insertion sizes (approximately 350 bp, 600 bp, 3 kb, and 10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (approximately 350× coverage) of data, the genome was assembled into 13,966 scaffolds with 50% of the assembly = 33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (approximately 153 kb) and a nearly complete mitochondrial genome (approximately 450 kb in 120 scaffolds). The nuclear genome is composed of 44,592 protein-coding genes. Genome resequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single-nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or -susceptible biotypes. The draft genome will be useful to better understand weediness and the evolution of herbicide resistance and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this article represents the first published draft genome of an agricultural weed.

  16. De novo assembly of transcriptome sequencing in Caragana korshinskii Kom. and characterization of EST-SSR markers.

    Directory of Open Access Journals (Sweden)

    Yan Long

    Full Text Available Caragana korshinskii Kom. is widely distributed in various habitats, including gravel desert, clay desert, fixed and semi-fixed sand, and saline land in the Asian and African deserts. To date, no previous genomic information or EST-SSR marker has been reported in Caragana Fabr. genus. In this study, more than two billion bases of high-quality sequence of C. korshinskii were generated by using illumina sequencing technology and demonstrated the de novo assembly and annotation of genes without prior genome information. These reads were assembled into 86,265 unigenes (mean length = 709 bp. The similarity search indicated that 33,955 and 21,978 unigenes showed significant similarities to known proteins from NCBI non-redundant and Swissprot protein databases, respectively. Among these annotated unigenes, 26,232 a unigenes were separately assigned to Gene Ontology (GO database. When 22,756 unigenes searched against the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG database, 5,598 unigenes were assigned to 5 main categories including 32 KEGG pathways. Among the main KEGG categories, metabolism was the biggest category (2,862, 43.7%, suggesting the active metabolic processes in the desert tree. In addition, a total of 19,150 EST-SSRs were identified from 15,484 unigenes, and the characterizations of EST-SSRs were further compared with other four species in Fabraceae. 126 potential marker sites were randomly selected to validate the assembly quality and develop EST-SSR markers. Among the 9 germplasms in Caranaga Fabr. genus, PCR success rate were 93.7% and the phylogenic tree was constructed based on the genotypic data. This research generated a substantial fraction of transcriptome sequences, which were very useful resources for gene annotation and discovery, molecular markers development, genome assembly and annotation. The EST-SSR markers identified and developed in this study will facilitate marker-assisted selection breeding.

  17. Optimization of de novo transcriptome assembly from high-throughput short read sequencing data improves functional annotation for non-model organisms

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    Haznedaroglu Berat Z

    2012-07-01

    Full Text Available Abstract Background The k-mer hash length is a key factor affecting the output of de novo transcriptome assembly packages using de Bruijn graph algorithms. Assemblies constructed with varying single k-mer choices might result in the loss of unique contiguous sequences (contigs and relevant biological information. A common solution to this problem is the clustering of single k-mer assemblies. Even though annotation is one of the primary goals of a transcriptome assembly, the success of assembly strategies does not consider the impact of k-mer selection on the annotation output. This study provides an in-depth k-mer selection analysis that is focused on the degree of functional annotation achieved for a non-model organism where no reference genome information is available. Individual k-mers and clustered assemblies (CA were considered using three representative software packages. Pair-wise comparison analyses (between individual k-mers and CAs were produced to reveal missing Kyoto Encyclopedia of Genes and Genomes (KEGG ortholog identifiers (KOIs, and to determine a strategy that maximizes the recovery of biological information in a de novo transcriptome assembly. Results Analyses of single k-mer assemblies resulted in the generation of various quantities of contigs and functional annotations within the selection window of k-mers (k-19 to k-63. For each k-mer in this window, generated assemblies contained certain unique contigs and KOIs that were not present in the other k-mer assemblies. Producing a non-redundant CA of k-mers 19 to 63 resulted in a more complete functional annotation than any single k-mer assembly. However, a fraction of unique annotations remained (~0.19 to 0.27% of total KOIs in the assemblies of individual k-mers (k-19 to k-63 that were not present in the non-redundant CA. A workflow to recover these unique annotations is presented. Conclusions This study demonstrated that different k-mer choices result in various quantities

  18. Transcriptome de novo assembly from next-generation sequencing and comparative analyses in the hexaploid salt marsh species Spartina maritima and Spartina alterniflora (Poaceae).

    Science.gov (United States)

    Ferreira de Carvalho, J; Poulain, J; Da Silva, C; Wincker, P; Michon-Coudouel, S; Dheilly, A; Naquin, D; Boutte, J; Salmon, A; Ainouche, M

    2013-02-01

    Spartina species have a critical ecological role in salt marshes and represent an excellent system to investigate recurrent polyploid speciation. Using the 454 GS-FLX pyrosequencer, we assembled and annotated the first reference transcriptome (from roots and leaves) for two related hexaploid Spartina species that hybridize in Western Europe, the East American invasive Spartina alterniflora and the Euro-African S. maritima. The de novo read assembly generated 38 478 consensus sequences and 99% found an annotation using Poaceae databases, representing a total of 16 753 non-redundant genes. Spartina expressed sequence tags were mapped onto the Sorghum bicolor genome, where they were distributed among the subtelomeric arms of the 10 S. bicolor chromosomes, with high gene density correlation. Normalization of the complementary DNA library improved the number of annotated genes. Ecologically relevant genes were identified among GO biological function categories in salt and heavy metal stress response, C4 photosynthesis and in lignin and cellulose metabolism. Expression of some of these genes had been found to be altered by hybridization and genome duplication in a previous microarray-based study in Spartina. As these species are hexaploid, up to three duplicated homoeologs may be expected per locus. When analyzing sequence polymorphism at four different loci in S. maritima and S. alterniflora, we found up to four haplotypes per locus, suggesting the presence of two expressed homoeologous sequences with one or two allelic variants each. This reference transcriptome will allow analysis of specific Spartina genes of ecological or evolutionary interest, estimation of homoeologous gene expression variation using RNA-seq and further gene expression evolution analyses in natural populations.

  19. Whole exome sequencing identifies de novo heterozygous CAV1 mutations associated with a novel neonatal onset lipodystrophy syndrome.

    Science.gov (United States)

    Garg, Abhimanyu; Kircher, Martin; Del Campo, Miguel; Amato, R Stephen; Agarwal, Anil K

    2015-08-01

    Despite remarkable progress in identifying causal genes for many types of genetic lipodystrophies in the last decade, the molecular basis of many extremely rare lipodystrophy patients with distinctive phenotypes remains unclear. We conducted whole exome sequencing of the parents and probands from six pedigrees with neonatal onset of generalized loss of subcutaneous fat with additional distinctive phenotypic features and report de novo heterozygous null mutations, c.424C>T (p.Q142*) and c.479_480delTT (p.F160*), in CAV1 in a 7-year-old male and a 3-year-old female of European origin, respectively. Both the patients had generalized fat loss, thin mottled skin and progeroid features at birth. The male patient had cataracts requiring extraction at age 30 months and the female patient had pulmonary arterial hypertension. Dermal fibroblasts of the female patient revealed negligible CAV1 immunofluorescence staining compared to control but there were no differences in the number and morphology of caveolae upon electron microscopy examination. Based upon the similarities in the clinical features of these two patients, previous reports of CAV1 mutations in patients with lipodystrophies and pulmonary hypertension, and similar features seen in CAV1 null mice, we conclude that these variants are the most likely cause of one subtype of neonatal onset generalized lipodystrophy syndrome.

  20. Characterization and analysis of a de novo transcriptome from the pygmy grasshopper Tetrix japonica.

    Science.gov (United States)

    Qiu, Zhongying; Liu, Fei; Lu, Huimeng; Huang, Yuan

    2016-06-11

    The pygmy grasshopper Tetrix japonica is a common insect distributed throughout the world, and it has the potential for use in studies of body colour polymorphism, genomics and the biology of Tetrigoidea (Insecta: Orthoptera). However, limited biological information is available for this insect. Here, we conducted a de novo transcriptome study of adult and larval T. japonica to provide a better understanding of its gene expression and develop genomic resources for future work. We sequenced and explored the characteristics of the de novo transcriptome of T. japonica using Illumina HiSeq 2000 platform. A total of 107 608 206 paired-end clean reads were assembled into 61 141 unigenes using the trinity software; the mean unigene size was 771 bp, and the N50 length was 1238 bp. A total of 29 225 unigenes were functionally annotated to the NCBI nonredundant protein sequences (Nr), NCBI nonredundant nucleotide sequences (Nt), a manually annotated and reviewed protein sequence database (Swiss-Prot), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of putative genes that are potentially involved in pigment pathways, juvenile hormone (JH) metabolism and signalling pathways were identified in the T. japonica transcriptome. Additionally, 165 769 and 156 796 putative single nucleotide polymorphisms occurred in the adult and larvae transcriptomes, respectively, and a total of 3162 simple sequence repeats were detected in this assembly. This comprehensive transcriptomic data for T. japonica will provide a usable resource for gene predictions, signalling pathway investigations and molecular marker development for this species and other pygmy grasshoppers.

  1. De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits.

    Science.gov (United States)

    Li, Ying-hui; Zhou, Guangyu; Ma, Jianxin; Jiang, Wenkai; Jin, Long-guo; Zhang, Zhouhao; Guo, Yong; Zhang, Jinbo; Sui, Yi; Zheng, Liangtao; Zhang, Shan-shan; Zuo, Qiyang; Shi, Xue-hui; Li, Yan-fei; Zhang, Wan-ke; Hu, Yiyao; Kong, Guanyi; Hong, Hui-long; Tan, Bing; Song, Jian; Liu, Zhang-xiong; Wang, Yaoshen; Ruan, Hang; Yeung, Carol K L; Liu, Jian; Wang, Hailong; Zhang, Li-juan; Guan, Rong-xia; Wang, Ke-jing; Li, Wen-bin; Chen, Shou-yi; Chang, Ru-zhen; Jiang, Zhi; Jackson, Scott A; Li, Ruiqiang; Qiu, Li-juan

    2014-10-01

    Wild relatives of crops are an important source of genetic diversity for agriculture, but their gene repertoire remains largely unexplored. We report the establishment and analysis of a pan-genome of Glycine soja, the wild relative of cultivated soybean Glycine max, by sequencing and de novo assembly of seven phylogenetically and geographically representative accessions. Intergenomic comparisons identified lineage-specific genes and genes with copy number variation or large-effect mutations, some of which show evidence of positive selection and may contribute to variation of agronomic traits such as biotic resistance, seed composition, flowering and maturity time, organ size and final biomass. Approximately 80% of the pan-genome was present in all seven accessions (core), whereas the rest was dispensable and exhibited greater variation than the core genome, perhaps reflecting a role in adaptation to diverse environments. This work will facilitate the harnessing of untapped genetic diversity from wild soybean for enhancement of elite cultivars.

  2. Evolution of the complement system in protostomes revealed by de novo transcriptome analysis of six species of Arthropoda.

    Science.gov (United States)

    Sekiguchi, Reo; Nonaka, Masaru

    2015-05-01

    To elucidate the evolutionary history of the complement system in Arthropoda, de novo transcriptome analysis was performed with six species among the Chelicerata, Myriapoda, and Crustacea, and complement genes were identified based on their characteristic domain structures. Complement C3 and factor B (FB) were identified from a sea spider, a jumping spider, and a centipede, but not from a sea firefly or two millipede species. No additional complement components identifiable by their characteristic domain structures were found from any of these six species. These results together with genome sequence information for several species of the Hexapoda suggest that the common ancestor of the Arthropoda possessed a simple complement system comprising C3 and FB, and thus resembled the alternative pathway of the mammalian complement system. It was lost at least twice independently during the evolution of Arthropoda in the millipede lineage and in the common ancestor of Crustacea and Hexapoda.

  3. Sequencing, de novo assembly and characterization of the spotted scat Scatophagus argus (Linnaeus 1766) transcriptome for discovery of reproduction related genes and SSRs

    Science.gov (United States)

    Yang, Wei; Chen, Huapu; Cui, Xuefan; Zhang, Kewei; Jiang, Dongneng; Deng, Siping; Zhu, Chunhua; Li, Guangli

    2017-09-01

    Spotted scat (Scatophagus argus) is an economically important farmed fish, particularly in East and Southeast Asia. Because there has been little research on reproductive development and regulation in this species, the lack of a mature artificial reproduction technology remains a barrier for the sustainable development of the aquaculture industry. More genetic and genomic background knowledge is urgently needed for an in-depth understanding of the molecular mechanism of reproductive process and identification of functional genes related to sexual differentiation, gonad maturation and gametogenesis. For these reasons, we performed transcriptomic analysis on spotted scat using a multiple tissue sample mixing strategy. The Illumina RNA sequencing generated 118 510 486 raw reads. After trimming, de novo assembly was performed and yielded 99 888 unigenes with an average length of 905.75 bp. A total of 45 015 unigenes were successfully annotated to the Nr, Swiss-Prot, KOG and KEGG databases. Additionally, 23 783 and 27 183 annotated unigenes were assigned to 56 Gene Ontology (GO) functional groups and 228 KEGG pathways, respectively. Subsequently, 2 474 transcripts associated with reproduction were selected using GO term and KEGG pathway assignments, and a number of reproduction-related genes involved in sex differentiation, gonad development and gametogenesis were identified. Furthermore, 22 279 simple sequence repeat (SSR) loci were discovered and characterized. The comprehensive transcript dataset described here greatly increases the genetic information available for spotted scat and contributes valuable sequence resources for functional gene mining and analysis. Candidate transcripts involved in reproduction would make good starting points for future studies on reproductive mechanisms, and the putative sex differentiation-related genes will be helpful for sex-determining gene identification and sex-specific marker isolation. Lastly, the SSRs can serve as marker

  4. Stable isotope N-phosphorylation labeling for Peptide de novo sequencing and protein quantification based on organic phosphorus chemistry.

    Science.gov (United States)

    Gao, Xiang; Wu, Hanzhi; Lee, Kim-Chung; Liu, Hongxia; Zhao, Yufen; Cai, Zongwei; Jiang, Yuyang

    2012-12-04

    In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ε-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents.

  5. Motor Sequence Learning and Consolidation in Unilateral De Novo Patients with Parkinson's Disease.

    Directory of Open Access Journals (Sweden)

    Xiaojuan Dan

    Full Text Available Previous research investigating motor sequence learning (MSL and consolidation in patients with Parkinson's disease (PD has predominantly included heterogeneous participant samples with early and advanced disease stages; thus, little is known about the onset of potential behavioral impairments. We employed a multisession MSL paradigm to investigate whether behavioral deficits in learning and consolidation appear immediately after or prior to the detection of clinical symptoms in the tested (left hand. Specifically, our patient sample was limited to recently diagnosed patients with pure unilateral PD. The left hand symptomatic (LH-S patients provided an assessment of performance following the onset of clinical symptoms in the tested hand. Conversely, right hand affected (left hand asymptomatic, LH-A patients served to investigate whether MSL impairments appear before symptoms in the tested hand. LH-S patients demonstrated impaired learning during the initial training session and both LH-S and LH-A patients demonstrated decreased performance compared to controls during the next-day retest. Critically, the impairments in later learning stages in the LH-A patients were evident even before the appearance of traditional clinical symptoms in the tested hand. Results may be explained by the progression of disease-related alterations in relevant corticostriatal networks.

  6. A De Novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny

    Science.gov (United States)

    Pucker, Boas; Holtgräwe, Daniela; Rosleff Sörensen, Thomas; Stracke, Ralf; Viehöver, Prisca

    2016-01-01

    Arabidopsis thaliana is the most important model organism for fundamental plant biology. The genome diversity of different accessions of this species has been intensively studied, for example in the 1001 genome project which led to the identification of many small nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). In addition, presence/absence variation (PAV), copy number variation (CNV) and mobile genetic elements contribute to genomic differences between A. thaliana accessions. To address larger genome rearrangements between the A. thaliana reference accession Columbia-0 (Col-0) and another accession of about average distance to Col-0, we created a de novo next generation sequencing (NGS)-based assembly from the accession Niederzenz-1 (Nd-1). The result was evaluated with respect to assembly strategy and synteny to Col-0. We provide a high quality genome sequence of the A. thaliana accession (Nd-1, LXSY01000000). The assembly displays an N50 of 0.590 Mbp and covers 99% of the Col-0 reference sequence. Scaffolds from the de novo assembly were positioned on the basis of sequence similarity to the reference. Errors in this automatic scaffold anchoring were manually corrected based on analyzing reciprocal best BLAST hits (RBHs) of genes. Comparison of the final Nd-1 assembly to the reference revealed duplications and deletions (PAV). We identified 826 insertions and 746 deletions in Nd-1. Randomly selected candidates of PAV were experimentally validated. Our Nd-1 de novo assembly allowed reliable identification of larger genic and intergenic variants, which was difficult or error-prone by short read mapping approaches alone. While overall sequence similarity as well as synteny is very high, we detected short and larger (affecting more than 100 bp) differences between Col-0 and Nd-1 based on bi-directional comparisons. The de novo assembly provided here and additional assemblies that will certainly be published in the future will allow to

  7. De novo transcriptome analysis of Perna viridis highlights tissue-specific patterns for environmental studies.

    Science.gov (United States)

    Leung, Priscilla T Y; Ip, Jack C H; Mak, Sarah S T; Qiu, Jian Wen; Lam, Paul K S; Wong, Chris K C; Chan, Leo L; Leung, Kenneth M Y

    2014-09-19

    The tropical green-lipped mussel Perna viridis is a common biomonitor throughout the Indo-Pacific region that is used for environmental monitoring and ecotoxicological investigations. However, there is limited molecular data available regarding this species. We sought to establish a global transcriptome database from the tissues of adductor muscle, gills and the hepatopancreas of P. viridis in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses in this sentinel species. Illumina sequencing results yielded 544,272,542 high-quality filtered reads. After de novo assembly using Trinity, 233,257 contigs were generated with an average length of 1,264 bp and an N50 length of 2,868 bp; 192,879 assembled transcripts and 150,111 assembled unigenes were obtained after clustering. A total of 93,668 assembled transcripts (66,692 assembled genes) with putative functions for protein domains were predicted based on InterProScan analysis. Based on similarity searches, 44,713 assembled transcripts and 25,319 assembled unigenes were annotated with at least one BLAST hit. A total of 21,262 assembled transcripts (11,947 assembled genes) were annotated with at least one well-defined Gene Ontology (GO) and 5,131 assembled transcripts (3,181 assembled unigenes) were assigned to 329 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The quantity of assembled unigenes and transcripts obtained from male and female mussels were similar but varied among the three studied tissues, with the highest numbers recorded in the gills, followed by the hepatopancreas, and then the adductor muscle. Multivariate analyses revealed strong tissue-specific patterns among the three different tissues, but not between sexes in terms of expression profiles for annotated genes in various GO terms, and genes associated with stress responses and degradation of xenobiotics. The expression profiles of certain selected genes in each tissue type were further

  8. De novo transcriptome sequence assembly from coconut leaves and seeds with a focus on factors involved in RNA-directed DNA methylation.

    Science.gov (United States)

    Huang, Ya-Yi; Lee, Chueh-Pai; Fu, Jason L; Chang, Bill Chia-Han; Matzke, Antonius J M; Matzke, Marjori

    2014-09-04

    Coconut palm (Cocos nucifera) is a symbol of the tropics and a source of numerous edible and nonedible products of economic value. Despite its nutritional and industrial significance, coconut remains under-represented in public repositories for genomic and transcriptomic data. We report de novo transcript assembly from RNA-seq data and analysis of gene expression in seed tissues (embryo and endosperm) and leaves of a dwarf coconut variety. Assembly of 10 GB sequencing data for each tissue resulted in 58,211 total unigenes in embryo, 61,152 in endosperm, and 33,446 in leaf. Within each unigene pool, 24,857 could be annotated in embryo, 29,731 could be annotated in endosperm, and 26,064 could be annotated in leaf. A KEGG analysis identified 138, 138, and 139 pathways, respectively, in transcriptomes of embryo, endosperm, and leaf tissues. Given the extraordinarily large size of coconut seeds and the importance of small RNA-mediated epigenetic regulation during seed development in model plants, we used homology searches to identify putative homologs of factors required for RNA-directed DNA methylation in coconut. The findings suggest that RNA-directed DNA methylation is important during coconut seed development, particularly in maturing endosperm. This dataset will expand the genomics resources available for coconut and provide a foundation for more detailed analyses that may assist molecular breeding strategies aimed at improving this major tropical crop.

  9. De novo sequencing of root transcriptome reveals complex cadmium-responsive regulatory networks in radish (Raphanus sativus L.).

    Science.gov (United States)

    Xu, Liang; Wang, Yan; Liu, Wei; Wang, Jin; Zhu, Xianwen; Zhang, Keyun; Yu, Rugang; Wang, Ronghua; Xie, Yang; Zhang, Wei; Gong, Yiqin; Liu, Liwang

    2015-07-01

    Cadmium (Cd) is a nonessential metallic trace element that poses potential chronic toxicity to living organisms. To date, little is known about the Cd-responsive regulatory network in root vegetable crops including radish. In this study, 31,015 unigenes representing 66,552 assembled unique transcripts were isolated from radish root under Cd stress based on de novo transcriptome assembly. In all, 1496 differentially expressed genes (DEGs) consisted of 3579 transcripts were identified from Cd-free (CK) and Cd-treated (Cd200) libraries. Gene Ontology and pathway enrichment analysis indicated that the up- and down-regulated DEGs were predominately involved in glucosinolate biosynthesis as well as cysteine and methionine-related pathways, respectively. RT-qPCR showed that the expression profiles of DEGs were in consistent with results from RNA-Seq analysis. Several candidate genes encoding phytochelatin synthase (PCS), metallothioneins (MTs), glutathione (GSH), zinc iron permease (ZIPs) and ABC transporter were responsible for Cd uptake, accumulation, translocation and detoxification in radish. The schematic model of DEGs and microRNAs-involved in Cd-responsive regulatory network was proposed. This study represents a first comprehensive transcriptome-based characterization of Cd-responsive DEGs in radish. These results could provide fundamental insight into complex Cd-responsive regulatory networks and facilitate further genetic manipulation of Cd accumulation in root vegetable crops.

  10. De novo sequencing of Hypericum perforatum transcriptome to identify potential genes involved in the biosynthesis of active metabolites.

    Directory of Open Access Journals (Sweden)

    Miao He

    Full Text Available BACKGROUND: Hypericum perforatum L. (St. John's wort is a medicinal plant with pharmacological properties that are antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial. Its major active metabolites are hypericins, hyperforins, and melatonin. However, little genetic information is available for this species, especially that concerning the biosynthetic pathways for active ingredients. METHODOLOGY/PRINCIPAL FINDINGS: Using de novo transcriptome analysis, we obtained 59,184 unigenes covering the entire life cycle of these plants. In all, 40,813 unigenes (68.86% were annotated and 2,359 were assigned to secondary metabolic pathways. Among them, 260 unigenes are involved in the production of hypericin, hyperforin, and melatonin. Another 2,291 unigenes are classified as potential Type III polyketide synthase. Our BlastX search against the AGRIS database reveals 1,772 unigenes that are homologous to 47 known Arabidopsis transcription factor families. Further analysis shows that 10.61% (6,277 of these unigenes contain 7,643 SSRs. CONCLUSION: We have identified a set of putative genes involved in several secondary metabolism pathways, especially those related to the synthesis of its active ingredients. Our results will serve as an important platform for public information about gene expression, genomics, and functional genomics in H. perforatum.

  11. De novo transcriptome sequencing to identify the sex-determination genes in Hyriopsis schlegelii.

    Science.gov (United States)

    Shi, Jianwu; Hong, Yijiang; Sheng, Junqing; Peng, Kou; Wang, Junhua

    2015-01-01

    This study presents the first analysis of expressed transcripts in the spermary and ovary of Hyriopsis schlegelii (H. schlegelii). A total of 132,055 unigenes were obtained and 31,781 of these genes were annotated. In addition, 19,511 upregulated and 25,911 downregulated unigenes were identified in the spermary. Ten sex-determination genes were selected and further analyzed by real-time PCR. In addition, mammalian genes reported to govern sex-determination pathways, including Sry, Dmrt1, Dmrt2, Sox9, GATA4, and WT1 in males and Wnt4, Rspo1, Foxl2, and β-catenin in females, were also identified in H. schlegelii. These results suggest that H. schlegelii and mammals use similar gene regulatory mechanisms to control sex determination. Moreover, genes associated with dosage compensation mechanisms, such as Msl1, Msl2, and Msl3, and hermaphrodite phenotypes, such as Tra-1, Tra-2α, Tra-2β, Fem1A, Fem1B, and Fem1C, were also identified in H. schlegelii. The identification of these genes indicates that diverse regulatory mechanisms regulate sexual polymorphism in H. schlegelii.

  12. Exome analysis of Smith-Magenis-like syndrome cohort identifies de novo likely pathogenic variants.

    Science.gov (United States)

    Berger, Seth I; Ciccone, Carla; Simon, Karen L; Malicdan, May Christine; Vilboux, Thierry; Billington, Charles; Fischer, Roxanne; Introne, Wendy J; Gropman, Andrea; Blancato, Jan K; Mullikin, James C; Gahl, William A; Huizing, Marjan; Smith, Ann C M

    2017-02-17

    Smith-Magenis syndrome (SMS), a neurodevelopmental disorder characterized by dysmorphic features, intellectual disability (ID), and sleep disturbances, results from a 17p11.2 microdeletion or a mutation in the RAI1 gene. We performed exome sequencing on 6 patients with SMS-like phenotypes but without chromosomal abnormalities or RAI1 variants. We identified pathogenic de novo variants in two cases, a nonsense variant in IQSEC2 and a missense variant in the SAND domain of DEAF1, and candidate de novo missense variants in an additional two cases. One candidate variant was located in an alpha helix of Necdin (NDN), phased to the paternally inherited allele. NDN is maternally imprinted within the 15q11.2 Prader-Willi Syndrome (PWS) region. This can help clarify NDN's role in the PWS phenotype. No definitive pathogenic gene variants were detected in the remaining SMS-like cases, but we report our findings for future comparison. This study provides information about the inheritance pattern and recurrence risk for patients with identified variants and demonstrates clinical and genetic overlap of neurodevelopmental disorders. Identification and characterization of ID-related genes that assist in development of common developmental pathways and/or gene-networks, may inform disease mechanism and treatment strategies.

  13. Fractals in DNA sequence analysis

    Institute of Scientific and Technical Information of China (English)

    Yu Zu-Guo(喻祖国); Vo Anh; Gong Zhi-Min(龚志民); Long Shun-Chao(龙顺潮)

    2002-01-01

    Fractal methods have been successfully used to study many problems in physics, mathematics, engineering, finance,and even in biology. There has been an increasing interest in unravelling the mysteries of DNA; for example, how can we distinguish coding and noncoding sequences, and the problems of classification and evolution relationship of organisms are key problems in bioinformatics. Although much research has been carried out by taking into consideration the long-range correlations in DNA sequences, and the global fractal dimension has been used in these works by other people, the models and methods are somewhat rough and the results are not satisfactory. In recent years, our group has introduced a time series model (statistical point of view) and a visual representation (geometrical point of view)to DNA sequence analysis. We have also used fractal dimension, correlation dimension, the Hurst exponent and the dimension spectrum (multifractal analysis) to discuss problems in this field. In this paper, we introduce these fractal models and methods and the results of DNA sequence analysis.

  14. De novo assembly and transcriptome analysis of osmoregulation in Litopenaeus vannamei under three cultivated conditions with different salinities.

    Science.gov (United States)

    Zhang, Dan; Wang, Fang; Dong, Shuanglin; Lu, Yunliang

    2016-03-10

    Litopenaeus vannamei, one of the most important euryhaline crustaceans, is cultured in seawater, brackish water, and freshwater worldwide. We performed Illumina RNA sequencing of L. vannamei gills, generating 124,914,870; 119,250,450; and 105,487,350 raw reads from the shrimps cultured in seawater, brackish water, and freshwater, respectively. From these reads, 466,293 transcripts were de novo assembled and annotated. Comparative genomic analysis showed that 1752 genes were significantly differentially expressed in the freshwater group compared with the seawater group, including 1242 upregulated and 510 downregulated genes. In addition, 1246 genes were differentially expressed in the brackish group vs. the seawater water group, including 659 upregulated and 587 downregulated genes. These differentially expressed genes were mainly involved in energy metabolism, substance metabolism, ion transport and signal transduction, and genetic process. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to analyze the functional significance of the differentially expressed genes, included those responding to salinity through diverse biological functions and processes and numerous potential genes associated with the osmotic response. L. vannamei responses to the three cultivated salinities were analyzed using next-generation sequencing. The transcriptional database established from the current research adds to the information available on L. vannamei and the findings expand our knowledge of the molecular basis of osmoregulation mechanisms in this species.

  15. How stable are Parkinson's disease subtypes in de novo patients: Analysis of the PPMI cohort?

    Science.gov (United States)

    Simuni, Tanya; Caspell-Garcia, Chelsea; Coffey, Christopher; Lasch, Shirley; Tanner, Caroline; Marek, Ken

    2016-07-01

    To determine the frequency and stability over time of the subgroup characterization of the tremor dominant (TD) versus postural instability gait disorder dominant (PIGD) Parkinson's disease (PD) in de novo patients. There is a substantial body of literature on the clinical sub classification of PD into TD versus PIGD subtype. However, there are limited data on the stability of this classification especially in early disease. Parkinson's Progression Markers Initiative (PPMI) is a longitudinal case control study of de novo, untreated PD participants at enrollment. Participants undergo a number of assessments including the Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS). TD versus PIGD subtype was defined based on the previously published formula. We report one-year analysis data. 320 of 423 PD recruited subjects had data on subtype classification at year 1 and were included in the analysis. 228 (71%) were classified as TD, 56 (18%) as PIGD and 36 (11%) as indeterminate at baseline. At 12 months, 39% PIGD and 18% TD shifted subtypes: 29% PIGD shifted to TD and 11% to Indeterminate; 10% TD shifted to PIGD and 8% to Indeterminate. The classification was not affected by the dopaminergic treatment (p = 0.59). TD versus PIGD subtype classification has substantial variability over first year in PD de novo cohort specifically for PIGD subtype. Dopaminergic therapy does not impact the change of the PD subtype. This instability has to be taken into consideration specifically when establishing correlations with the biomarkers and for long term prognostication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Integrated sequence analysis. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Andersson, K.; Pyy, P

    1998-02-01

    The NKS/RAK subprojet 3 `integrated sequence analysis` (ISA) was formulated with the overall objective to develop and to test integrated methodologies in order to evaluate event sequences with significant human action contribution. The term `methodology` denotes not only technical tools but also methods for integration of different scientific disciplines. In this report, we first discuss the background of ISA and the surveys made to map methods in different application fields, such as man machine system simulation software, human reliability analysis (HRA) and expert judgement. Specific event sequences were, after the surveys, selected for application and testing of a number of ISA methods. The event sequences discussed in the report were cold overpressure of BWR, shutdown LOCA of BWR, steam generator tube rupture of a PWR and BWR disturbed signal view in the control room after an external event. Different teams analysed these sequences by using different ISA and HRA methods. Two kinds of results were obtained from the ISA project: sequence specific and more general findings. The sequence specific results are discussed together with each sequence description. The general lessons are discussed under a separate chapter by using comparisons of different case studies. These lessons include areas ranging from plant safety management (design, procedures, instrumentation, operations, maintenance and safety practices) to methodological findings (ISA methodology, PSA,HRA, physical analyses, behavioural analyses and uncertainty assessment). Finally follows a discussion about the project and conclusions are presented. An interdisciplinary study of complex phenomena is a natural way to produce valuable and innovative results. This project came up with structured ways to perform ISA and managed to apply the in practice. The project also highlighted some areas where more work is needed. In the HRA work, development is required for the use of simulators and expert judgement as

  17. Structural and functional analysis of PUR2,5 gene encoding bifunctional enzyme of de novo purine biosynthesis in Ogataea (Hansenula) polymorpha CBS 4732T.

    Science.gov (United States)

    Stoyanov, Anton; Petrova, Penka; Lyutskanova, Dimitrinka; Lahtchev, Kantcho

    2014-01-01

    We describe the cloning, sequencing and functional characterization of gene PUR2,5, involved in de novo purine biosynthesis of the yeast Ogataea (Hansenula) polymorpha. This gene (2369 bp) was cloned by genetic complementation of adenine requiring mutation. It encodes a bifunctional enzyme of 789 amino acids (85 kDa) that catalyzes the second and the fifth steps of de novo purine biosynthesis pathway and shows dual enzymatic activity - of glycinamide ribotide synthetase (GARS, EC 6.3.4.13) and of aminoimidazole ribotide synthetase (AIRS, EC 6.3.3.1). Nucleotide sequence analysis revealed the presence of putative regulatory elements located in the adjacent 5' region. Canonical motives that function as binding sites for BAS1 transcription activator were found at positions (-593) and (-389). The putative TAATTA-box was located at (-20) to (-14) and AT-rich heteroduplex was found in the 3'-non-translated region. We compared the amino acid sequence of OpPUR2,5p with those of the corresponding enzymes of other yeast species as well as with distant organisms like bacteria Escherichia coli and human Homo sapiens. A successful disruption of OpPUR2,5 gene was done. It was found that OpPUR2,5::LEU2 replacement affects both mating and sporulation processes. OpPUR2,5 sequence is deposited in the GenBank of NCBI with accession no. JF967633. Copyright © 2013 Elsevier GmbH. All rights reserved.

  18. De novo transcriptome assembly analysis of weed Apera spica-venti from seven tissues and growth stages.

    Science.gov (United States)

    Babineau, Marielle; Mahmood, Khalid; Mathiassen, Solvejg K; Kudsk, Per; Kristensen, Michael

    2017-02-06

    Loose silky bentgrass (Apera spica-venti) is an important weed in Europe with a recent increase in herbicide resistance cases. The lack of genetic information about this noxious weed limits its biological understanding such as growth, reproduction, genetic variation, molecular ecology and metabolic herbicide resistance. This study produced a reference transcriptome for A. spica-venti from different tissues (leaf, root, stem) and various growth stages (seed at phenological stages 05, 07, 08, 09). The de novo assembly was performed on individual and combined dataset followed by functional annotations. Individual transcripts and gene families involved in metabolic based herbicide resistance were identified. Eight separate transcriptome assemblies were performed and compared. The combined transcriptome assembly consists of 83,349 contigs with an N50 and average contig length of 762 and 658 bp, respectively. This dataset contains 74,724 transcripts consisting of total 54,846,111 bp. Among them 94% had a homologue to UniProtKB, 73% retrieved a GO mapping, and 50% were functionally annotated. Compared with other grass species, A. spica-venti has 26% proteins in common to Brachypodium distachyon, and 41% to Lolium spp. Glycosyltransferases had the highest number of transcripts in each tissue followed by the cytochrome P450s. The GSTF1 and CYP89A2 transcripts were recovered from the majority of tissues and aligned at a maximum of 66 and 30% to proven herbicide resistant allele from Alopecurus myosuroides and Lolium rigidum, respectively. De novo transcriptome assembly enabled the generation of the first reference transcriptome of A. spica-venti. This can serve as stepping stone for understanding the metabolic herbicide resistance as well as the general biology of this problematic weed. Furthermore, this large-scale sequence data is a valuable scientific resource for comparative transcriptome analysis for Poaceae grasses.

  19. Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11 kb), single molecule, real-time sequencing.

    Science.gov (United States)

    Vembar, Shruthi Sridhar; Seetin, Matthew; Lambert, Christine; Nattestad, Maria; Schatz, Michael C; Baybayan, Primo; Scherf, Artur; Smith, Melissa Laird

    2016-08-01

    The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed AT-richness [∼80.6% (A + T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12 kb, with 50% of the reads between 15.5 and 50 kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [∼90-99% (A + T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission.

  20. De novo assembly and transcriptome analysis of two contrary tillering mutants to learn the mechanisms of tillers outgrowth in switchgrass (Panicum virgatum L.

    Directory of Open Access Journals (Sweden)

    Kaijie eXu

    2015-09-01

    Full Text Available Tillering is an important trait in monocotyledon plants. The switchgrass (Panicum virgatum, studied usually as a source of biomass for energy production, can produce hundreds of tillers in its lifetime. Studying the tillering of switchgrass also provides information for other monocot crops. High-tillering and low-tillering mutants were produced by ethyl methanesulfonate (EMS mutagenesis. Alteration of tillering ability resulted from different tiller buds outgrowth in the two mutants. We sequenced the tiller buds transcriptomes of high-tillering and low-tillering plants using next-generation sequencing technology, and generated 34 G data in total. In the de novo assembly results, 133,828 unigenes were detected with an average length of 1,238 bp, and 5,290 unigenes were differentially expressed between the two mutants, including 3,225 up-regulated genes and 2,065 down-regulated genes. Differentially expressed gene (DEG analysis with functional annotations was performed to identify candidate genes involved in tiller bud outgrowth processes using Gene Ontology (GO classification, Cluster of Orthologous Groups of proteins (COG, and Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analysis. This is the first study to explore the tillering transcriptome in two types of tillering mutants by de novo sequencing.

  1. Sequence Handling by Sequence Analysis Toolbox v1.0

    DEFF Research Database (Denmark)

    Ingrell, Christian Ravnsborg; Matthiesen, Rune; Jensen, Ole Nørregaard

    2006-01-01

    The fact that mass spectrometry have become a high-throughput method calls for bioinformatic tools for automated sequence handling and prediction. For efficient use of bioinformatic tools, it is important that these tools are integrated or interfaced with each other. The purpose of sequence...... analysis toolbox v1.0 was to have a general purpose sequence analyzing tool that can import sequences obtained by high-throughput sequencing methods. The program includes algorithms for calculation or prediction of isoelectric point, hydropathicity index, transmembrane segments, and glycosylphosphatidyl...

  2. Transcriptome Profile of the Asian Giant Hornet (Vespa mandarinia) Using Illumina HiSeq 4000 Sequencing: De Novo Assembly, Functional Annotation, and Discovery of SSR Markers

    OpenAIRE

    Bharat Bhusan Patnaik; So Young Park; Se Won Kang; Hee-Ju Hwang; Tae Hun Wang; Eun Bi Park; Jong Min Chung; Dae Kwon Song; Changmu Kim; Soonok Kim; Jae Bong Lee; Heon Cheon Jeong; Hong Seog Park; Yeon Soo Han; Yong Seok Lee

    2016-01-01

    Vespa mandarinia found in the forests of East Asia, including Korea, occupies the highest rank in the arthropod food web within its geographical range. It serves as a source of nutrition in the form of Vespa amino acid mixture and is listed as a threatened species, although no conservation measures have been implemented. Here, we performed de novo assembly of the V. mandarinia transcriptome by Illumina HiSeq 4000 sequencing. Over 60 million raw reads and 59,184,811 clean reads were obtained. ...

  3. Tracing the evolutionary lineage of pattern recognition receptor homologues in vertebrates: An insight into reptilian immunity via de novo sequencing of the wall lizard splenic transcriptome.

    Science.gov (United States)

    Priyam, Manisha; Tripathy, Mamta; Rai, Umesh; Ghorai, Soma Mondal

    2016-04-01

    Reptiles remain a deprived class in the area of genomic and molecular resources for the vertebrate classes. The transition of squamates from aquatic to terrestrial mode of life caused profound changes in their immune system to combat the altered variety of pathogens on land. The current study aims at delineating the evolution of defence mechanisms in wall lizard, Hemidactylus flaviviridis, by exploring its immunome. De novo sequencing of splenic transcriptome from wall lizard on the Illumina Hi-Seq platform generated 258,128 unique transcripts with an average GC content of 45%. Annotation of 555,557 and 6812 transcripts was carried out against NCBI (non-redundant database) and UniProt databases, respectively. The KEGG pathway annotation of transcripts classified them into 39 processes of six pathway function categories. A total of 3824 transcripts, involved in 23 immune-related pathways, were identified in the immune-relevant cluster built by harvesting the genes under KEGG pathways of immune system and immune diseases. Forty-two percent of the immune-relevant cluster was represented by pattern-recognition receptors (PRRs), of which the maximum number of transcripts was attributed to the Toll-like receptor (TLR) signalling pathway. Nine PRRs with potential full-length coding sequences were sorted for phylogenetic analysis and comparative domain analysis across the vertebrate lineage. They included DEC205/lymphocyte antigen 75 (ly75), nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), NOD-like receptor family CARD domain-containing 3 (NLRC3), nucleotide-binding oligomerisation domain, leucine-rich repeat-containing X1 (NLRX1), DDX58/retinoic acid-inducible gene 1 (RIG-1), Toll-like receptor 3 (TLR3), TLR4, TLR5 and TLR7. From selection studies of these genes, we inferred positive selection for ly75, NOD1, RIG-1, TLR3 and TLR4. Apart from contributing to the scarce genomic resources available for reptiles and giving a broad scope for the immune

  4. De Novo Assembly and Transcriptome Analysis of Bulb Onion (Allium cepa L.) during Cold Acclimation Using Contrasting Genotypes.

    Science.gov (United States)

    Han, Jeongsukhyeon; Thamilarasan, Senthil Kumar; Natarajan, Sathishkumar; Park, Jong-In; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Bulb onion (Allium cepa) is the second most widely cultivated and consumed vegetable crop in the world. During winter, cold injury can limit the production of bulb onion. Genomic resources available for bulb onion are still very limited. To date, no studies on heritably durable cold and freezing tolerance have been carried out in bulb onion genotypes. We applied high-throughput sequencing technology to cold (2°C), freezing (-5 and -15°C), and control (25°C)-treated samples of cold tolerant (CT) and cold susceptible (CS) genotypes of A. cepa lines. A total of 452 million paired-end reads were de novo assembled into 54,047 genes with an average length of 1,331 bp. Based on similarity searches, these genes were aligned with entries in the public non-redundant (nr) database, as well as KEGG and COG database. Differentially expressed genes (DEGs) were identified using log10 values with the FPKM method. Among 5,167DEGs, 491 genes were differentially expressed at freezing temperature compared to the control temperature in both CT and CS libraries. The DEG results were validated with qRT-PCR. We performed GO and KEGG pathway enrichment analyses of all DEGs and iPath interactive analysis found 31 pathways including those related to metabolism of carbohydrate, nucleotide, energy, cofactors and vitamins, other amino acids and xenobiotics biodegradation. Furthermore, a large number of molecular markers were identified from the assembled genes, including simple sequence repeats (SSRs) 4,437 and SNP substitutions of transition and transversion types of CT and CS. Our study is the first to provide a transcriptome sequence resource for Allium spp. with regard to cold and freezing stress. We identified a large set of genes and determined their DEG profiles under cold and freezing conditions using two different genotypes. These data represent a valuable resource for genetic and genomic studies of Allium spp.

  5. Sequence Analysis in Demographic Research

    Directory of Open Access Journals (Sweden)

    Billari, Francesco C.

    2001-01-01

    Full Text Available EnglishThis paper examines the salient features of sequence analysis in demogrpahicresearch. The new approach allows a holistic perspective on life course analysis and is based on arepresentation of lives as sequences of states. Some of the methods for analyzing such data aresketched, from complex description to optimal matching ot monoethetic divisive algorithms. Afer ashort ilustration of a demographically-relevant example, the needs in terms of data collection and theopportunities of applying the same aproach to synthetic data are discussed.FrenchOn examine ici les principaux éléments de l’analyse par séquence endémographie. Cette nouvelle technique permet une perspective unifiée del’analyse du cours de la vie, en représentant la vie comme une série d’états.Certaines des méthodes pour de telles analyses sont décrites, en commençant parla description complexe, pour considérer ensuite les alignements optimales, etles algorithmes de division. Après un court exemple en démographie, onconsidère les besoins en données et les possibilités d’application aux donnéessynthétique.

  6. Hunting down frame shifts: Ecological analysis of diverse functional gene sequences

    Directory of Open Access Journals (Sweden)

    Michal eStrejcek

    2015-11-01

    Full Text Available Functional gene ecological analyses using amplicon sequencing can be challenging as translated sequences are often burdened with shifted reading frames. The aim of this work was to evaluate several bioinformatics tools designed to correct errors which arise during sequencing in an effort to reduce the number of frame-shifts (FS. Genes encoding for alpha subunits of biphenyl (bphA and benzoate (benA dioxygenases were used as model sequences. FrameBot, a FS correction tool, was able to reduce the number of detected FS to zero. However, up to 43.1% of sequences were discarded by FrameBot as non-specific targets. Therefore, we proposed a de novo mode of FrameBot for FS correction, which works on a similar basis as common chimera identifying platforms and is not dependent on reference sequences. By nature of FrameBot de novo design, it is crucial to provide it with data as error free as possible. We tested the ability of several publicly available correction tools to decrease the number of errors in the data sets. The combination of Maximum Expected Error (MEE filtering and single linkage pre-clustering (SLP proved the most efficient read procession. Applying FrameBot de novo on the processed data enabled analysis of BphA sequences with minimal losses of potentially functional sequences not homologous to those previously known. This experiment also demonstrated the extensive diversity of dioxygenases in soil. A script which performs FrameBot de novo is presented in the supplementary material to the study and the tool was implemented into FunGene Pipeline available at http://fungene.cme.msu.edu/FunGenePipeline/ and https://github.com/rdpstaff/Framebot.

  7. Detection of a Usp-like gene in Calotropis procera plant from the de novo assembled genome contigs of the high-throughput sequencing dataset.

    Science.gov (United States)

    Shokry, Ahmed M; Al-Karim, Saleh; Ramadan, Ahmed; Gadallah, Nour; Al Attas, Sanaa G; Sabir, Jamal S M; Hassan, Sabah M; Madkour, Magdy A; Bressan, Ray; Mahfouz, Magdy; Bahieldin, Ahmed

    2014-02-01

    The wild plant species Calotropis procera (C. procera) has many potential applications and beneficial uses in medicine, industry and ornamental field. It also represents an excellent source of genes for drought and salt tolerance. Genes encoding proteins that contain the conserved universal stress protein (USP) domain are known to provide organisms like bacteria, archaea, fungi, protozoa and plants with the ability to respond to a plethora of environmental stresses. However, information on the possible occurrence of Usp in C. procera is not available. In this study, we uncovered and characterized a one-class A Usp-like (UspA-like, NCBI accession No. KC954274) gene in this medicinal plant from the de novo assembled genome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for Usp sequences were blasted with the recovered de novo assembled contigs. Homology modelling of the deduced amino acids (NCBI accession No. AGT02387) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera USPA-like full sequence model on Thermus thermophilus USP UniProt protein (PDB accession No. Q5SJV7) was constructed using RasMol and Deep-View programs. The functional domains of the novel USPA-like amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).

  8. Detection of a Usp-like gene in Calotropis procera plant from the de novo assembled genome contigs of the high-throughput sequencing dataset

    KAUST Repository

    Shokry, Ahmed M.

    2014-02-01

    The wild plant species Calotropis procera (C. procera) has many potential applications and beneficial uses in medicine, industry and ornamental field. It also represents an excellent source of genes for drought and salt tolerance. Genes encoding proteins that contain the conserved universal stress protein (USP) domain are known to provide organisms like bacteria, archaea, fungi, protozoa and plants with the ability to respond to a plethora of environmental stresses. However, information on the possible occurrence of Usp in C. procera is not available. In this study, we uncovered and characterized a one-class A Usp-like (UspA-like, NCBI accession No. KC954274) gene in this medicinal plant from the de novo assembled genome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for Usp sequences were blasted with the recovered de novo assembled contigs. Homology modelling of the deduced amino acids (NCBI accession No. AGT02387) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera USPA-like full sequence model on Thermus thermophilus USP UniProt protein (PDB accession No. Q5SJV7) was constructed using RasMol and Deep-View programs. The functional domains of the novel USPA-like amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM). © 2014 Académie des sciences.

  9. De novo transcriptome analysis and molecular marker development of two Hemarthria species

    Directory of Open Access Journals (Sweden)

    Xiu eHuang

    2016-04-01

    Full Text Available Hemarthria R. Br. is an important genus of perennial forage grasses that is widely used in subtropical and tropical regions. Hemarthria grasses have made remarkable contributions to the development of animal husbandry and agro-ecosystem maintenance; however, there is currently a lack of comprehensive genomic data available for these species. In this study, we used Illumina high-throughput deep sequencing to characterize of two agriculturally important Hemarthria materials, H. compressa ‘Yaan’ and H. altissima ‘1110.’ Sequencing runs that used each of four normalized RNA samples from the leaves or roots of the two materials yielded more than 24 million high-quality reads. After de novo assembly, 137,142 and 77,150 unigenes were obtained for ‘Yaan’ and ‘1110’, respectively. In addition, a total of 86,731 ‘Yaan’ and 48,645 ‘1110’ unigenes were successfully annotated. After consolidating the unigenes for both materials, 42,646 high-quality SNPs were identified in 10,880 unigenes and 10,888 SSRs were identified in 8,330 unigenes. To validate the identified markers, high quality PCR primers were designed for both SNPs and SSRs. We randomly tested 16 of the SNP primers and 54 of the SSR primers and found that the majority of these primers successfully amplified the desired PCR product. In addition, high cross-species transferability (61.11%-87.04% of SSR markers was achieved for four other Poaceae species. The amount of RNA sequencing data that was generated for these two Hemarthria species greatly increases the amount of genomic information available for Hemarthria and the SSR and SNP markers identified in this study will facilitate further advancements in genetic and molecular studies of the Hemarthria genus.

  10. Sequencing and de novo assembly of the Asian clam (Corbicula fluminea transcriptome using the Illumina GAIIx method.

    Directory of Open Access Journals (Sweden)

    Huihui Chen

    Full Text Available BACKGROUND: The Asian clam (Corbicula fluminea is currently one of the most economically important aquatic species in China and has been used as a test organism in many environmental studies. However, the lack of genomic resources, such as sequenced genome, expressed sequence tags (ESTs and transcriptome sequences has hindered the research on C. fluminea. Recent advances in large-scale RNA-Seq enable generation of genomic resources in a short time, and provide large expression datasets for functional genomic analysis. METHODOLOGY/PRINCIPAL FINDINGS: We used a next-generation high-throughput DNA sequencing technique with an Illumina GAIIx method to analyze the transcriptome from the whole bodies of C. fluminea. More than 62,250,336 high-quality reads were generated based on the raw data, and 134,684 unigenes with a mean length of 791 bp were assembled using the Velvet and Oases software. All of the assembly unigenes were annotated by running BLASTx and BLASTn similarity searches on the Nt, Nr, Swiss-Prot, COG and KEGG databases. In addition, the Clusters of Orthologous Groups (COGs, Gene Ontology (GO terms and Kyoto Encyclopedia of Gene and Genome (KEGG annotations were also assigned to each unigene transcript. To provide a preliminary verification of the assembly and annotation results, and search for potential environmental pollution biomarkers, 15 functional genes (five antioxidase genes, two cytochrome P450 genes, three GABA receptor-related genes and five heat shock protein genes were cloned and identified. Expressions of the 15 selected genes following fluoxetine exposure confirmed that the genes are indeed linked to environmental stress. CONCLUSIONS/SIGNIFICANCE: The C. fluminea transcriptome advances the underlying molecular understanding of this freshwater clam, provides a basis for further exploration of C. fluminea as an environmental test organism and promotes further studies on other bivalve organisms.

  11. De novo transcriptome and small RNA analysis of two Chinese willow cultivars reveals stress response genes in Salix matsudana.

    Science.gov (United States)

    Rao, Guodong; Sui, Jinkai; Zeng, Yanfei; He, Caiyun; Duan, Aiguo; Zhang, Jianguo

    2014-01-01

    Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar 'Tortuosa'). De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs) and 36 different expressed miRNAs (DEMs). Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix.

  12. RNA-Seq analysis using de novo transcriptome assembly as a reference for the salmon louse Caligus rogercresseyi.

    Directory of Open Access Journals (Sweden)

    Cristian Gallardo-Escárate

    Full Text Available Despite the economic and environmental impacts that sea lice infestations have on salmon farming worldwide, genomic data generated by high-throughput transcriptome sequencing for different developmental stages, sexes, and strains of sea lice is still limited or unknown. In this study, RNA-seq analysis was performed using de novo transcriptome assembly as a reference for evidenced transcriptional changes from six developmental stages of the salmon louse Caligus rogercresseyi. EST-datasets were generated from the nauplius I, nauplius II, copepodid and chalimus stages and from female and male adults using MiSeq Illumina sequencing. A total of 151,788,682 transcripts were yielded, which were assembled into 83,444 high quality contigs and subsequently annotated into roughly 24,000 genes based on known proteins. To identify differential transcription patterns among salmon louse stages, cluster analyses were performed using normalized gene expression values. Herein, four clusters were differentially expressed between nauplius I-II and copepodid stages (604 transcripts, five clusters between copepodid and chalimus stages (2,426 transcripts, and six clusters between female and male adults (2,478 transcripts. Gene ontology analysis revealed that the nauplius I-II, copepodid and chalimus stages are mainly annotated to aminoacid transfer/repair/breakdown, metabolism, molting cycle, and nervous system development. Additionally, genes showing differential transcription in female and male adults were highly related to cytoskeletal and contractile elements, reproduction, cell development, morphogenesis, and transcription-translation processes. The data presented in this study provides the most comprehensive transcriptome resource available for C. rogercresseyi, which should be used for future genomic studies linked to host-parasite interactions.

  13. De novo transcriptome and small RNA analysis of two Chinese willow cultivars reveals stress response genes in Salix matsudana.

    Directory of Open Access Journals (Sweden)

    Guodong Rao

    Full Text Available Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar 'Tortuosa'. De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs and 36 different expressed miRNAs (DEMs. Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix.

  14. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    Science.gov (United States)

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  15. Information Analysis of DNA Sequences

    CERN Document Server

    Mohammed, Riyazuddin

    2010-01-01

    The problem of differentiating the informational content of coding (exons) and non-coding (introns) regions of a DNA sequence is one of the central problems of genomics. The introns are estimated to be nearly 95% of the DNA and since they do not seem to participate in the process of transcription of amino-acids, they have been termed "junk DNA." Although it is believed that the non-coding regions in genomes have no role in cell growth and evolution, demonstration that these regions carry useful information would tend to falsify this belief. In this paper, we consider entropy as a measure of information by modifying the entropy expression to take into account the varying length of these sequences. Exons are usually much shorter in length than introns; therefore the comparison of the entropy values needs to be normalized. A length correction strategy was employed using randomly generated nucleonic base strings built out of the alphabet of the same size as the exons under question. Our analysis shows that intron...

  16. De novo assembly and transcriptome analysis of the Mediterranean fruit fly Ceratitis capitata early embryos.

    Directory of Open Access Journals (Sweden)

    Marco Salvemini

    Full Text Available The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, belongs to the Tephritidae family, which includes a large number of other damaging pest species. The Medfly has been the first non-drosophilid fly species which has been genetically transformed paving the way for designing genetic-based pest control strategies. Furthermore, it is an experimentally tractable model, in which transient and transgene-mediated RNAi have been successfully used. We applied Illumina sequencing to total RNA preparations of 8-10 hours old embryos of C. capitata, This developmental window corresponds to the blastoderm cellularization stage. In summary, we assembled 42,614 transcripts which cluster in 26,319 unique transcripts of which 11,045 correspond to protein coding genes; we identified several hundreds of long ncRNAs; we found an enrichment of transcripts encoding RNA binding proteins among the highly expressed transcripts, such as CcTRA-2, known to be necessary to establish and, most likely, to maintain female sex of C. capitata. Our study is the first de novo assembly performed for Ceratitis capitata based on Illumina NGS technology during embryogenesis and it adds novel data to the previously published C. capitata EST databases. We expect that it will be useful for a variety of applications such as gene cloning and phylogenetic analyses, as well as to advance genetic research and biotechnological applications in the Medfly and other related Tephritidae.

  17. De novo assembly and transcriptome analysis of the Mediterranean fruit fly Ceratitis capitata early embryos.

    Science.gov (United States)

    Salvemini, Marco; Arunkumar, Kallare P; Nagaraju, Javaregowda; Sanges, Remo; Petrella, Valeria; Tomar, Archana; Zhang, Hongyu; Zheng, Weiwei; Saccone, Giuseppe

    2014-01-01

    The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, belongs to the Tephritidae family, which includes a large number of other damaging pest species. The Medfly has been the first non-drosophilid fly species which has been genetically transformed paving the way for designing genetic-based pest control strategies. Furthermore, it is an experimentally tractable model, in which transient and transgene-mediated RNAi have been successfully used. We applied Illumina sequencing to total RNA preparations of 8-10 hours old embryos of C. capitata, This developmental window corresponds to the blastoderm cellularization stage. In summary, we assembled 42,614 transcripts which cluster in 26,319 unique transcripts of which 11,045 correspond to protein coding genes; we identified several hundreds of long ncRNAs; we found an enrichment of transcripts encoding RNA binding proteins among the highly expressed transcripts, such as CcTRA-2, known to be necessary to establish and, most likely, to maintain female sex of C. capitata. Our study is the first de novo assembly performed for Ceratitis capitata based on Illumina NGS technology during embryogenesis and it adds novel data to the previously published C. capitata EST databases. We expect that it will be useful for a variety of applications such as gene cloning and phylogenetic analyses, as well as to advance genetic research and biotechnological applications in the Medfly and other related Tephritidae.

  18. Specific versus non-specific immune responses in an invertebrate species evidenced by a comparative de novo sequencing study.

    Directory of Open Access Journals (Sweden)

    Emeline Deleury

    Full Text Available Our present understanding of the functioning and evolutionary history of invertebrate innate immunity derives mostly from studies on a few model species belonging to ecdysozoa. In particular, the characterization of signaling pathways dedicated to specific responses towards fungi and Gram-positive or Gram-negative bacteria in Drosophila melanogaster challenged our original view of a non-specific immunity in invertebrates. However, much remains to be elucidated from lophotrochozoan species. To investigate the global specificity of the immune response in the fresh-water snail Biomphalaria glabrata, we used massive Illumina sequencing of 5'-end cDNAs to compare expression profiles after challenge by Gram-positive or Gram-negative bacteria or after a yeast challenge. 5'-end cDNA sequencing of the libraries yielded over 12 millions high quality reads. To link these short reads to expressed genes, we prepared a reference transcriptomic database through automatic assembly and annotation of the 758,510 redundant sequences (ESTs, mRNAs of B. glabrata available in public databases. Computational analysis of Illumina reads followed by multivariate analyses allowed identification of 1685 candidate transcripts differentially expressed after an immune challenge, with a two fold ratio between transcripts showing a challenge-specific expression versus a lower or non-specific differential expression. Differential expression has been validated using quantitative PCR for a subset of randomly selected candidates. Predicted functions of annotated candidates (approx. 700 unisequences belonged to a large extend to similar functional categories or protein types. This work significantly expands upon previous gene discovery and expression studies on B. glabrata and suggests that responses to various pathogens may involve similar immune processes or signaling pathways but different genes belonging to multigenic families. These results raise the question of the importance

  19. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  20. Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.

    Science.gov (United States)

    Shevchenko, A; Chernushevich, I; Ens, W; Standing, K G; Thomson, B; Wilm, M; Mann, M

    1997-01-01

    Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts. While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories 'de novo' protein sequencing is still performed by Edman degradation. Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides. Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization. A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument. Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000. Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach. The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms. The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.

  1. Rapid genome mapping in nanochannel arrays for highly complete and accurate de novo sequence assembly of the complex Aegilops tauschii genome.

    Science.gov (United States)

    Hastie, Alex R; Dong, Lingli; Smith, Alexis; Finklestein, Jeff; Lam, Ernest T; Huo, Naxin; Cao, Han; Kwok, Pui-Yan; Deal, Karin R; Dvorak, Jan; Luo, Ming-Cheng; Gu, Yong; Xiao, Ming

    2013-01-01

    Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs assembly from 75% to 95% complete.

  2. Rapid genome mapping in nanochannel arrays for highly complete and accurate de novo sequence assembly of the complex Aegilops tauschii genome.

    Directory of Open Access Journals (Sweden)

    Alex R Hastie

    Full Text Available Next-generation sequencing (NGS technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum. Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.

  3. A multi-platform draft de novo genome assembly and comparative analysis for the Scarlet Macaw (Ara macao).

    Science.gov (United States)

    Seabury, Christopher M; Dowd, Scot E; Seabury, Paul M; Raudsepp, Terje; Brightsmith, Donald J; Liboriussen, Poul; Halley, Yvette; Fisher, Colleen A; Owens, Elaine; Viswanathan, Ganesh; Tizard, Ian R

    2013-01-01

    Data deposition to NCBI Genomes: This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMXX00000000 (SMACv1.0, unscaffolded genome assembly). The version described in this paper is the first version (AMXX01000000). The scaffolded assembly (SMACv1.1) has been deposited at DDBJ/EMBL/GenBank under the accession AOUJ00000000, and is also the first version (AOUJ01000000). Strong biological interest in traits such as the acquisition and utilization of speech, cognitive abilities, and longevity catalyzed the utilization of two next-generation sequencing platforms to provide the first-draft de novo genome assembly for the large, new world parrot Ara macao (Scarlet Macaw). Despite the challenges associated with genome assembly for an outbred avian species, including 951,507 high-quality putative single nucleotide polymorphisms, the final genome assembly (>1.035 Gb) includes more than 997 Mb of unambiguous sequence data (excluding N's). Cytogenetic analyses including ZooFISH revealed complex rearrangements associated with two scarlet macaw macrochromosomes (AMA6, AMA7), which supports the hypothesis that translocations, fusions, and intragenomic rearrangements are key factors associated with karyotype evolution among parrots. In silico annotation of the scarlet macaw genome provided robust evidence for 14,405 nuclear gene annotation models, their predicted transcripts and proteins, and a complete mitochondrial genome. Comparative analyses involving the scarlet macaw, chicken, and zebra finch genomes revealed high levels of nucleotide-based conservation as well as evidence for overall genome stability among the three highly divergent species. Application of a new whole-genome analysis of divergence involving all three species yielded prioritized candidate genes and noncoding regions for parrot traits of interest (i.e., speech, intelligence, longevity) which were independently supported by the results of previous human GWAS studies. We

  4. Nonlinear analysis of biological sequences

    Energy Technology Data Exchange (ETDEWEB)

    Torney, D.C.; Bruno, W.; Detours, V. [and others

    1998-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The main objectives of this project involved deriving new capabilities for analyzing biological sequences. The authors focused on tabulating the statistical properties exhibited by Human coding DNA sequences and on techniques of inferring the phylogenetic relationships among protein sequences related by descent.

  5. Gait Analysis by Multi Video Sequence Analysis

    DEFF Research Database (Denmark)

    Jensen, Karsten; Juhl, Jens

    2009-01-01

    The project presented in this article aims to develop software so that close-range photogrammetry with sufficient accuracy can be used to point out the most frequent foot mal positions and monitor the effect of the traditional treatment. The project is carried out as a cooperation between the Ort...... Sequence Analysis (MVSA). Results show that the developed MVSA system, in the following called Fodex, can measure the navicula height with a precision of 0.5-0.8 mm. The calcaneus angle can be measured with a precision of 0.8-1.5 degrees....

  6. De novo transcriptome assembly of Ipomoea nil using Illumina sequencing for gene discovery and SSR marker identification.

    Science.gov (United States)

    Wei, Changhe; Tao, Xiang; Li, Ming; He, Bin; Yan, Lang; Tan, Xuemei; Zhang, Yizheng

    2015-10-01

    Ipomoea nil is widely used as an ornamental plant due to its abundance of flower color, but the limited transcriptome and genomic data hinder research on it. Using illumina platform, transcriptome profiling of I. nil was performed through high-throughput sequencing, which was proven to be a rapid and cost-effective means to characterize gene content. Our goal is to use the resulting information to facilitate the relevant research on flowering and flower color formation in I. nil. In total, 268 million unique illumina RNA-Seq reads were produced and used in the transcriptome assembly. These reads were assembled into 220,117 contigs, of which 137,307 contigs were annotated using the GO and KEGG database. Based on the result of functional annotations, a total of 89,781 contigs were assigned 455,335 GO term annotations. Meanwhile, 17,418 contigs were identified with pathway annotation and they were functionally assigned to 144 KEGG pathways. Our transcriptome revealed at least 55 contigs as probably flowering-related genes in I. nil, and we also identified 25 contigs that encode key enzymes in the phenylpropanoid biosynthesis pathway. Based on the analysis relating to gene expression profiles, in the phenylpropanoid biosynthesis pathway of I. nil, the repression of lignin biosynthesis might lead to the redirection of the metabolic flux into anthocyanin biosynthesis. This may be the most likely reason that I. nil has high anthocyanins content, especially in its flowers. Additionally, 15,537 simple sequence repeats (SSRs) were detected using the MISA software, and these SSRs will undoubtedly benefit future breeding work. Moreover, the information uncovered in this study will also serve as a valuable resource for understanding the flowering and flower color formation mechanisms in I. nil.

  7. De novo computational identification of stress-related sequence motifs and microRNA target sites in untranslated regions of a plant translatome

    Science.gov (United States)

    Munusamy, Prabhakaran; Zolotarov, Yevgen; Meteignier, Louis-Valentin; Moffett, Peter; Strömvik, Martina V.

    2017-01-01

    Gene regulation at the transcriptional and translational level leads to diversity in phenotypes and function in organisms. Regulatory DNA or RNA sequence motifs adjacent to the gene coding sequence act as binding sites for proteins that in turn enable or disable expression of the gene. Whereas the known DNA and RNA binding proteins range in the thousands, only a few motifs have been examined. In this study, we have predicted putative regulatory motifs in groups of untranslated regions from genes regulated at the translational level in Arabidopsis thaliana under normal and stressed conditions. The test group of sequences was divided into random subgroups and subjected to three de novo motif finding algorithms (Seeder, Weeder and MEME). In addition to identifying sequence motifs, using an in silico tool we have predicted microRNA target sites in the 3′ UTRs of the translationally regulated genes, as well as identified upstream open reading frames located in the 5′ UTRs. Our bioinformatics strategy and the knowledge generated contribute to understanding gene regulation during stress, and can be applied to disease and stress resistant plant development. PMID:28276452

  8. Use of targeted next-generation sequencing for molecular diagnosis of craniosynostosis: Identification of a novel de novo mutation of EFNB1.

    Science.gov (United States)

    Yamamoto, Toshiyuki; Igarashi, Naru; Shimojima, Keiko; Sangu, Noriko; Sakamoto, Yuko; Shimoji, Kazuaki; Niijima, Shinichi

    2016-03-01

    Craniofrontonasal syndrome (CFNS; MIM#304110) is characterized by asymmetric facial features with hypertelorism and a broad bifid nose due to synostosis of the coronal suture. CFNS shows a unique X-linked inheritance pattern (most affected patients are female and obligate male carriers exhibit a mild manifestation or no typical features at all) associated with the ephrin-B1 gene (EFNB1) located in the Xq13.1 region. In this study, we performed targeted, massively parallel sequencing using a next-generation sequencer, and identified a novel EFNB1 mutation, c.270_271delCA, in a Japanese female patient with craniosynostosis. Because subsequent Sanger sequencing identified no mutation in either parent, this mutation was determined to be de novo in origin. After obtaining molecular diagnosis, a retrospective clinical evaluation confirmed the clinical diagnosis of CFNS in this patient. Comprehensive molecular diagnosis using a next-generation sequencer would be beneficial for early diagnosis of the patients with undiagnosed craniosynostosis.

  9. Bayesian analysis of binary sequences

    Science.gov (United States)

    Torney, David C.

    2005-03-01

    This manuscript details Bayesian methodology for "learning by example", with binary n-sequences encoding the objects under consideration. Priors prove influential; conformable priors are described. Laplace approximation of Bayes integrals yields posterior likelihoods for all n-sequences. This involves the optimization of a definite function over a convex domain--efficiently effectuated by the sequential application of the quadratic program.

  10. Joint Sequence Analysis: Association and Clustering

    Science.gov (United States)

    Piccarreta, Raffaella

    2017-01-01

    In its standard formulation, sequence analysis aims at finding typical patterns in a set of life courses represented as sequences. Recently, some proposals have been introduced to jointly analyze sequences defined on different domains (e.g., work career, partnership, and parental histories). We introduce measures to evaluate whether a set of…

  11. Exome sequencing identifies de novo pathogenic variants in FBN1 and TRPS1 in a patient with a complex connective tissue phenotype

    Science.gov (United States)

    Zastrow, Diane B.; Zornio, Patricia A.; Dries, Annika; Kohler, Jennefer; Fernandez, Liliana; Waggott, Daryl; Walkiewicz, Magdalena; Eng, Christine M.; Manning, Melanie A.; Farrelly, Ellyn; Fisher, Paul G.; Ashley, Euan A.; Bernstein, Jonathan A.

    2017-01-01

    Here we describe a patient who presented with a history of congenital diaphragmatic hernia, inguinal hernia, and recurrent umbilical hernia. She also has joint laxity, hypotonia, and dysmorphic features. A unifying diagnosis was not identified based on her clinical phenotype. As part of her evaluation through the Undiagnosed Diseases Network, trio whole-exome sequencing was performed. Pathogenic variants in FBN1 and TRPS1 were identified as causing two distinct autosomal dominant conditions, each with de novo inheritance. Fibrillin 1 (FBN1) mutations are associated with Marfan syndrome and a spectrum of similar phenotypes. TRPS1 mutations are associated with trichorhinophalangeal syndrome types I and III. Features of both conditions are evident in the patient reported here. Discrepant features of the conditions (e.g., stature) and the young age of the patient may have made a clinical diagnosis more difficult in the absence of exome-wide genetic testing. PMID:28050602

  12. De novo analysis of electron impact mass spectra using fragmentation trees

    Energy Technology Data Exchange (ETDEWEB)

    Hufsky, Franziska, E-mail: franziska.hufsky@uni-jena.de [Chair of Bioinformatics, Friedrich Schiller University, Ernst-Abbe-Platz 2, Jena (Germany); Max Planck Institute for Chemical Ecology, Beutenberg Campus, Jena (Germany); Rempt, Martin [Institute for Inorganic and Analytical Chemistry, Bioorganic Analytics, Friedrich Schiller University, Lessingstrasse 8, Jena (Germany); Rasche, Florian [Chair of Bioinformatics, Friedrich Schiller University, Ernst-Abbe-Platz 2, Jena (Germany); Pohnert, Georg [Institute for Inorganic and Analytical Chemistry, Bioorganic Analytics, Friedrich Schiller University, Lessingstrasse 8, Jena (Germany); Boecker, Sebastian, E-mail: sebastian.boecker@uni-jena.de [Chair of Bioinformatics, Friedrich Schiller University, Ernst-Abbe-Platz 2, Jena (Germany)

    2012-08-20

    Highlights: Black-Right-Pointing-Pointer We present a method for de novo analysis of accurate mass EI mass spectra of small molecules. Black-Right-Pointing-Pointer This method identifies the molecular ion and thus the molecular formula where the molecular ion is present in the spectrum. Black-Right-Pointing-Pointer Fragmentation trees are constructed by automated signal extraction and evaluation. Black-Right-Pointing-Pointer These trees explain relevant fragmentation reactions. Black-Right-Pointing-Pointer This method will be very helpful in the automated analysis of unknown metabolites. - Abstract: The automated fragmentation analysis of high resolution EI mass spectra based on a fragmentation tree algorithm is introduced. Fragmentation trees are constructed from EI spectra by automated signal extraction and evaluation. These trees explain relevant fragmentation reactions and assign molecular formulas to fragments. The method enables the identification of the molecular ion and the molecular formula of a metabolite if the molecular ion is present in the spectrum. These identifications are independent of existing library knowledge and, thus, support assignment and structural elucidation of unknown compounds. The method works even if the molecular ion is of very low abundance or hidden under contaminants with higher masses. We apply the algorithm to a selection of 50 derivatized and underivatized metabolites and demonstrate that in 78% of cases the molecular ion can be correctly assigned. The automatically constructed fragmentation trees correspond very well to published mechanisms and allow the assignment of specific relevant fragments and fragmentation pathways even in the most complex EI-spectra in our dataset. This method will be very helpful in the automated analysis of metabolites that are not included in common libraries and it thus has the potential to support the explorative character of metabolomics studies.

  13. De Novo Transcriptome Sequencing of the Octopus vulgaris Hemocytes Using Illumina RNA-Seq Technology: Response to the Infection by the Gastrointestinal Parasite Aggregata octopiana

    Science.gov (United States)

    Castellanos-Martínez, Sheila; Arteta, David; Catarino, Susana; Gestal, Camino

    2014-01-01

    Background Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus’ well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. Results A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. Conclusion The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers

  14. De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

    Directory of Open Access Journals (Sweden)

    Sheila Castellanos-Martínez

    Full Text Available BACKGROUND: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. RESULTS: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. CONCLUSION: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to

  15. Prenatal diagnosis and molecular cytogenetic analysis of a de novo isodicentric chromosome 18

    Science.gov (United States)

    Zhang, Yanliang; Dai, Yong; Ren, Jinghui; Wang, Linqian

    2010-01-01

    Isodicentric chromosome 18 [idic(18)] is rare structural aberration. We report on a prenatal case described by conventional and molecular cytogenetic analyses. The sonography at 24 weeks of gestation revealed multiple fetal anomalies; radial aplasia and ventricular septal defect were significant features. Routine karyotyping showed a derivative chromosome replacing one normal chromosome 18. The parental karyotypes were normal, indicating that the derivative chromosome was de novo. Array comparative genomic hybridization (array-CGH) revealed 18p11.21→qter duplication and 18p11.21→pter deletion for genomic DNA of the fetus. The breakpoint was located at 18p11.21 (between 12104527 bp and 12145199 bp from the telomere of 18p). Thus, the derivative chromosome was ascertained as idic(18)(qter→p11.21::p11.21→qter). Fluorescent in situ hybridization (FISH) confirmed that the derivative chromosome was idic(18). Our report describes a rare isodicentric chromosome 18 and demonstrates that array-CGH is a useful complementary tool to cytogenetic analysis for reliable identifying derivative chromosome. PMID:20864786

  16. De novo Transcriptome Analysis of Sinapis alba in Revealing the Glucosinolate and Phytochelatin Pathways

    Science.gov (United States)

    Zhang, Xiaohui; Liu, Tongjin; Duan, Mengmeng; Song, Jiangping; Li, Xixiang

    2016-01-01

    Sinapis alba is an important condiment crop and can also be used as a phytoremediation plant. Though it has important economic and agronomic values, sequence data, and the genetic tools are still rare in this plant. In the present study, a de novo transcriptome based on the transcriptions of leaves, stems, and roots was assembled for S. alba for the first time. The transcriptome contains 47,972 unigenes with a mean length of 1185 nt and an N50 of 1672 nt. Among these unigenes, 46,535 (97%) unigenes were annotated by at least one of the following databases: NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO), and Clusters of Orthologous Groups of proteins (COGs). The tissue expression pattern profiles revealed that 3489, 1361, and 8482 unigenes were predominantly expressed in the leaves, stems, and roots of S. alba, respectively. Genes predominantly expressed in the leaf were enriched in photosynthesis- and carbon fixation-related pathways. Genes predominantly expressed in the stem were enriched in not only pathways related to sugar, ether lipid, and amino acid metabolisms but also plant hormone signal transduction and circadian rhythm pathways, while the root-dominant genes were enriched in pathways related to lignin and cellulose syntheses, involved in plant-pathogen interactions, and potentially responsible for heavy metal chelating, and detoxification. Based on this transcriptome, 14,727 simple sequence repeats (SSRs) were identified, and 12,830 pairs of primers were developed for 2522 SSR-containing unigenes. Additionally, the glucosinolate (GSL) and phytochelatin metabolic pathways, which give the characteristic flavor and the heavy metal tolerance of this plant, were intensively analyzed. The genes of aliphatic GSLs pathway were predominantly expressed in roots. The absence of aliphatic GSLs in leaf tissues was due to the shutdown of BCAT4, MAM1, and CYP79F1 expressions. Glutathione was extensively

  17. De novo transcriptome analysis of Sinapis alba in revealing the glucosinolate and phytochelatin pathways

    Directory of Open Access Journals (Sweden)

    Xiaohui eZhang

    2016-03-01

    Full Text Available Sinapis alba is an important condiment crop and can also be used as a phytoremediation plant. Though it has important economic and agronomic values, sequence data and the genetic tools are still rare in this plant. In the present study, a de novo transcriptome based on the transcriptions of leaves, stems and roots was assembled for S. alba for the first time. The transcriptome contains 47,972 unigenes with a mean length of 1,185 nt and an N50 of 1,672 nt. Among these unigenes, 46,535 (97% unigenes were annotated by at least one of the following databases: NCBI non-redundant (Nr, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG pathway, Gene Ontology (GO, and Clusters of Orthologous Groups of proteins (COGs. The tissue expression pattern profiles revealed that 3,489, 1,361 and 8,482 unigenes were predominantly expressed in the leaves, stems and roots of S. alba, respectively. Genes predominantly expressed in the leaf were enriched in photosynthesis- and carbon fixation-related pathways. Genes predominantly expressed in the stem were enriched in not only pathways related to sugar, ether lipid and amino acid metabolisms but also plant hormone signal transduction and circadian rhythm pathways, while the root-dominant genes were enriched in pathways related to lignin and cellulose syntheses, involved in plant-pathogen interactions, and potentially responsible for heavy metal chelating and detoxification. Based on this transcriptome, 14,727 simple sequence repeats (SSRs were identified, and 12,830 pairs of primers were developed for 2,522 SSR-containing unigenes. Additionally, the glucosinolate (GSL and phytochelatin metabolic pathways, which give the characteristic flavor and the heavy metal tolerance of this plant, were intensively analyzed. The genes of aliphatic GSLs pathway were predominantly expressed in roots. The absence of aliphatic GSLs in leaf tissues was due to the shutdown of BCAT4, MAM1 and CYP79F1 expressions. Glutathione was

  18. De novo transcriptomic analysis of hydrogen production in the green alga Chlamydomonas moewusii through RNA-Seq.

    Science.gov (United States)

    Yang, Shihui; Guarnieri, Michael T; Smolinski, Sharon; Ghirardi, Maria; Pienkos, Philip T

    2013-08-23

    Microalgae can make a significant contribution towards meeting global renewable energy needs in both carbon-based and hydrogen (H2) biofuel. The development of energy-related products from algae could be accelerated with improvements in systems biology tools, and recent advances in sequencing technology provide a platform for enhanced transcriptomic analyses. However, these techniques are still heavily reliant upon available genomic sequence data. Chlamydomonas moewusii is a unicellular green alga capable of evolving molecular H2 under both dark and light anaerobic conditions, and has high hydrogenase activity that can be rapidly induced. However, to date, there is no systematic investigation of transcriptomic profiling during induction of H2 photoproduction in this organism. In this work, RNA-Seq was applied to investigate transcriptomic profiles during the dark anaerobic induction of H2 photoproduction. 156 million reads generated from 7 samples were then used for de novo assembly after data trimming. BlastX results against NCBI database and Blast2GO results were used to interpret the functions of the assembled 34,136 contigs, which were then used as the reference contigs for RNA-Seq analysis. Our results indicated that more contigs were differentially expressed during the period of early and higher H2 photoproduction, and fewer contigs were differentially expressed when H2-photoproduction rates decreased. In addition, C. moewusii and C. reinhardtii share core functional pathways, and transcripts for H2 photoproduction and anaerobic metabolite production were identified in both organisms. C. moewusii also possesses similar metabolic flexibility as C. reinhardtii, and the difference between C. moewusii and C. reinhardtii on hydrogenase expression and anaerobic fermentative pathways involved in redox balancing may explain their different profiles of hydrogenase activity and secreted anaerobic metabolites. Herein, we have described a workflow using commercial

  19. De Novo Assembly of Coding Sequences of the Mangrove Palm (Nypa fruticans Using RNA-Seq and Discovery of Whole-Genome Duplications in the Ancestor of Palms.

    Directory of Open Access Journals (Sweden)

    Ziwen He

    Full Text Available Nypa fruticans (Arecaceae is the only monocot species of true mangroves. This species represents the earliest mangrove fossil recorded. How N. fruticans adapts to the harsh and unstable intertidal zone is an interesting question. However, the 60 gene segments deposited in NCBI are insufficient for solving this question. In this study, we sequenced, assembled and annotated the transcriptome of N. fruticans using next-generation sequencing technology. A total of 19,918,800 clean paired-end reads were de novo assembled into 45,368 unigenes with a N50 length of 1,096 bp. A total of 41.35% unigenes were functionally annotated using Blast2GO. Many genes annotated to "response to stress" and 15 putative positively selected genes were identified. Simple sequence repeats were identified and compared with other palms. The divergence time between N. fruticans and other palms was estimated at 75 million years ago using the genomic data, which is consistent with the fossil record. After calculating the synonymous substitution rate between paralogs, we found that two whole-genome duplication events were shared by N. fruticans and other palms. These duplication events provided a large amount of raw material for the more than 2,000 later speciation events in Arecaceae. This study provides a high quality resource for further functional and evolutionary studies of N. fruticans and palms in general.

  20. De Novo Assembly of Coding Sequences of the Mangrove Palm (Nypa fruticans) Using RNA-Seq and Discovery of Whole-Genome Duplications in the Ancestor of Palms.

    Science.gov (United States)

    He, Ziwen; Zhang, Zhang; Guo, Wuxia; Zhang, Ying; Zhou, Renchao; Shi, Suhua

    2015-01-01

    Nypa fruticans (Arecaceae) is the only monocot species of true mangroves. This species represents the earliest mangrove fossil recorded. How N. fruticans adapts to the harsh and unstable intertidal zone is an interesting question. However, the 60 gene segments deposited in NCBI are insufficient for solving this question. In this study, we sequenced, assembled and annotated the transcriptome of N. fruticans using next-generation sequencing technology. A total of 19,918,800 clean paired-end reads were de novo assembled into 45,368 unigenes with a N50 length of 1,096 bp. A total of 41.35% unigenes were functionally annotated using Blast2GO. Many genes annotated to "response to stress" and 15 putative positively selected genes were identified. Simple sequence repeats were identified and compared with other palms. The divergence time between N. fruticans and other palms was estimated at 75 million years ago using the genomic data, which is consistent with the fossil record. After calculating the synonymous substitution rate between paralogs, we found that two whole-genome duplication events were shared by N. fruticans and other palms. These duplication events provided a large amount of raw material for the more than 2,000 later speciation events in Arecaceae. This study provides a high quality resource for further functional and evolutionary studies of N. fruticans and palms in general.

  1. De novo transcriptome assembly and comparative analysis of differentially expressed genes in Prunus dulcis Mill. in response to freezing stress.

    Directory of Open Access Journals (Sweden)

    Sadegh Mousavi

    Full Text Available Almond (Prunus dulcis Mill., one of the most important nut crops, requires chilling during winter to develop fruiting buds. However, early spring chilling and late spring frost may damage the reproductive tissues leading to reduction in the rate of productivity. Despite the importance of transcriptional changes and regulation, little is known about the almond's transcriptome under the cold stress conditions. In the current research, we used RNA-seq technique to study the response of the reproductive tissues of almond (anther and ovary to frost stress. RNA sequencing resulted in more than 20 million reads from anther and ovary tissues of almond, individually. About 40,000 contigs were assembled and annotated de novo in each tissue. Profile of gene expression in ovary showed significant alterations in 5,112 genes, whereas in anther 6,926 genes were affected by freezing stress. Around two thousands of these genes were common altered genes in both ovary and anther libraries. Gene ontology indicated the involvement of differentially expressed (DE genes, responding to freezing stress, in metabolic and cellular processes. qRT-PCR analysis verified the expression pattern of eight genes randomly selected from the DE genes. In conclusion, the almond gene index assembled in this study and the reported DE genes can provide great insights on responses of almond and other Prunus species to abiotic stresses. The obtained results from current research would add to the limited available information on almond and Rosaceae. Besides, the findings would be very useful for comparative studies as the number of DE genes reported here is much higher than that of any previous reports in this plant.

  2. Sequencing and comparative analysis of the gorilla MHC genomic sequence.

    Science.gov (United States)

    Wilming, Laurens G; Hart, Elizabeth A; Coggill, Penny C; Horton, Roger; Gilbert, James G R; Clee, Chris; Jones, Matt; Lloyd, Christine; Palmer, Sophie; Sims, Sarah; Whitehead, Siobhan; Wiley, David; Beck, Stephan; Harrow, Jennifer L

    2013-01-01

    Major histocompatibility complex (MHC) genes play a critical role in vertebrate immune response and because the MHC is linked to a significant number of auto-immune and other diseases it is of great medical interest. Here we describe the clone-based sequencing and subsequent annotation of the MHC region of the gorilla genome. Because the MHC is subject to extensive variation, both structural and sequence-wise, it is not readily amenable to study in whole genome shotgun sequence such as the recently published gorilla genome. The variation of the MHC also makes it of evolutionary interest and therefore we analyse the sequence in the context of human and chimpanzee. In our comparisons with human and re-annotated chimpanzee MHC sequence we find that gorilla has a trimodular RCCX cluster, versus the reference human bimodular cluster, and additional copies of Class I (pseudo)genes between Gogo-K and Gogo-A (the orthologues of HLA-K and -A). We also find that Gogo-H (and Patr-H) is coding versus the HLA-H pseudogene and, conversely, there is a Gogo-DQB2 pseudogene versus the HLA-DQB2 coding gene. Our analysis, which is freely available through the VEGA genome browser, provides the research community with a comprehensive dataset for comparative and evolutionary research of the MHC.

  3. IVA: accurate de novo assembly of RNA virus genomes.

    Science.gov (United States)

    Hunt, Martin; Gall, Astrid; Ong, Swee Hoe; Brener, Jacqui; Ferns, Bridget; Goulder, Philip; Nastouli, Eleni; Keane, Jacqueline A; Kellam, Paul; Otto, Thomas D

    2015-07-15

    An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and polymerase chain reaction amplification process of current methods. We developed a new de novo assembler called IVA (Iterative Virus Assembler) designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from human immunodeficiency virus-1 or influenza-virus-infected people and demonstrated that IVA outperforms all other virus de novo assemblers. The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/iva © The Author 2015. Published by Oxford University Press.

  4. The power of single molecule real-time sequencing technology in the de novo assembly of a eukaryotic genome.

    Science.gov (United States)

    Sakai, Hiroaki; Naito, Ken; Ogiso-Tanaka, Eri; Takahashi, Yu; Iseki, Kohtaro; Muto, Chiaki; Satou, Kazuhito; Teruya, Kuniko; Shiroma, Akino; Shimoji, Makiko; Hirano, Takashi; Itoh, Takeshi; Kaga, Akito; Tomooka, Norihiko

    2015-11-30

    Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. However, a large portion of the genomes still remains unassembled. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. The SMRT-based assembly produced 100 times longer contigs with 100 times smaller amount of gaps compared to the SGS-based assemblies. A detailed comparison between the assemblies revealed that the SMRT-based assembly enabled a more comprehensive gene annotation than the SGS-based assemblies where thousands of genes were missing or fragmented. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. We demonstrated that SMRT technology, though still needed support of SGS data, achieved a near-complete assembly of a eukaryotic genome.

  5. Fast, cheap and out of control--Insights into thermodynamic and informatic constraints on natural protein sequences from de novo protein design.

    Science.gov (United States)

    Brisendine, Joseph M; Koder, Ronald L

    2016-05-01

    The accumulated results of thirty years of rational and computational de novo protein design have taught us important lessons about the stability, information content, and evolution of natural proteins. First, de novo protein design has complicated the assertion that biological function is equivalent to biological structure - demonstrating the capacity to abstract active sites from natural contexts and paste them into non-native topologies without loss of function. The structure-function relationship has thus been revealed to be either a generality or strictly true only in a local sense. Second, the simplification to "maquette" topologies carried out by rational protein design also has demonstrated that even sophisticated functions such as conformational switching, cooperative ligand binding, and light-activated electron transfer can be achieved with low-information design approaches. This is because for simple topologies the functional footprint in sequence space is enormous and easily exceeds the number of structures which could have possibly existed in the history of life on Earth. Finally, the pervasiveness of extraordinary stability in designed proteins challenges accepted models for the "marginal stability" of natural proteins, suggesting that there must be a selection pressure against highly stable proteins. This can be explained using recent theories which relate non-equilibrium thermodynamics and self-replication. This article is part of a Special Issue entitled Biodesign for Bioenergetics--The design and engineering of electronc transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Whole exome sequencing is necessary to clarify ID/DD cases with de novo copy number variants of uncertain significance: Two proof-of-concept examples.

    Science.gov (United States)

    Giorgio, Elisa; Ciolfi, Andrea; Biamino, Elisa; Caputo, Viviana; Di Gregorio, Eleonora; Belligni, Elga Fabia; Calcia, Alessandro; Gaidolfi, Elena; Bruselles, Alessandro; Mancini, Cecilia; Cavalieri, Simona; Molinatto, Cristina; Cirillo Silengo, Margherita; Ferrero, Giovanni Battista; Tartaglia, Marco; Brusco, Alfredo

    2016-07-01

    Whole exome sequencing (WES) is a powerful tool to identify clinically undefined forms of intellectual disability/developmental delay (ID/DD), especially in consanguineous families. Here we report the genetic definition of two sporadic cases, with syndromic ID/DD for whom array-Comparative Genomic Hybridization (aCGH) identified a de novo copy number variant (CNV) of uncertain significance. The phenotypes included microcephaly with brachycephaly and a distinctive facies in one proband, and hypotonia in the legs and mild ataxia in the other. WES allowed identification of a functionally relevant homozygous variant affecting a known disease gene for rare syndromic ID/DD in each proband, that is, c.1423C>T (p.Arg377*) in the Trafficking Protein Particle Complex 9 (TRAPPC9), and c.154T>C (p.Cys52Arg) in the Very Low Density Lipoprotein Receptor (VLDLR). Four mutations affecting TRAPPC9 have been previously reported, and the present finding further depicts this syndromic form of ID, which includes microcephaly with brachycephaly, corpus callosum hypoplasia, facial dysmorphism, and overweight. VLDLR-associated cerebellar hypoplasia (VLDLR-CH) is characterized by non-progressive congenital ataxia and moderate-to-profound intellectual disability. The c.154T>C (p.Cys52Arg) mutation was associated with a very mild form of ataxia, mild intellectual disability, and cerebellar hypoplasia without cortical gyri simplification. In conclusion, we report two novel cases with rare causes of autosomal recessive ID, which document how interpreting de novo array-CGH variants represents a challenge in consanguineous families; as such, clinical WES should be considered in diagnostic testing. © 2016 Wiley Periodicals, Inc.

  7. Massively parallel sequencing reveals an accumulation of de novo mutations and an activating mutation of LPAR1 in a patient with metastatic neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Jun S Wei

    Full Text Available Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy. Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1 had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.

  8. A multi-platform draft de novo genome assembly and comparative analysis for the Scarlet Macaw (Ara macao.

    Directory of Open Access Journals (Sweden)

    Christopher M Seabury

    Full Text Available Data deposition to NCBI Genomes: This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMXX00000000 (SMACv1.0, unscaffolded genome assembly. The version described in this paper is the first version (AMXX01000000. The scaffolded assembly (SMACv1.1 has been deposited at DDBJ/EMBL/GenBank under the accession AOUJ00000000, and is also the first version (AOUJ01000000. Strong biological interest in traits such as the acquisition and utilization of speech, cognitive abilities, and longevity catalyzed the utilization of two next-generation sequencing platforms to provide the first-draft de novo genome assembly for the large, new world parrot Ara macao (Scarlet Macaw. Despite the challenges associated with genome assembly for an outbred avian species, including 951,507 high-quality putative single nucleotide polymorphisms, the final genome assembly (>1.035 Gb includes more than 997 Mb of unambiguous sequence data (excluding N's. Cytogenetic analyses including ZooFISH revealed complex rearrangements associated with two scarlet macaw macrochromosomes (AMA6, AMA7, which supports the hypothesis that translocations, fusions, and intragenomic rearrangements are key factors associated with karyotype evolution among parrots. In silico annotation of the scarlet macaw genome provided robust evidence for 14,405 nuclear gene annotation models, their predicted transcripts and proteins, and a complete mitochondrial genome. Comparative analyses involving the scarlet macaw, chicken, and zebra finch genomes revealed high levels of nucleotide-based conservation as well as evidence for overall genome stability among the three highly divergent species. Application of a new whole-genome analysis of divergence involving all three species yielded prioritized candidate genes and noncoding regions for parrot traits of interest (i.e., speech, intelligence, longevity which were independently supported by the results of previous human GWAS

  9. Sequencing, annotation and comparative analysis of nine BACs of giant panda (Ailuropoda melanoleuca).

    Science.gov (United States)

    Zheng, Yang; Cai, Jing; Li, JianWen; Li, Bo; Lin, RunMao; Tian, Feng; Wang, XiaoLing; Wang, Jun

    2010-01-01

    A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.

  10. Sequencing,annotation and comparative analysis of nine BACs of the giant panda(Ailuropoda melanoleuca)

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A 10-fold BAC library for the giant panda was constructed and nine BACs were selected to generate finish sequences.These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of the giant panda newly generated by Illumina GA sequencing technology.Complete Sanger sequencing,assembly,annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb.Homologue search and de novo prediction methods were used to annotate genes and repeats.Twelve protein coding genes were predicted,seven of which could be functionally annotated.The seven genes have an average gene size of about 41 kb,an average coding size of about 1.2 kb and an average exon number of 6 per gene.Besides,seven tRNA genes were found.About 27 percent of the BAC sequence is composed of repeats.A phylogenetic tree was constructed using a neighbor-join algorithm across five species,including the giant panda,human,dog,cat and mouse,which reconfirms dog as the most closely related species to the giant panda.Our results provide detailed sequence and structure information for new genes and repeats of the giant panda,which will be helpful for further studies about the giant panda.

  11. De Novo Transcriptomic Analysis of an Oleaginous Microalga: Pathway Description and Gene Discovery for Production of Next-Generation Biofuels

    Science.gov (United States)

    Wan, LingLin; Han, Juan; Sang, Min; Li, AiFen; Wu, Hong; Yin, ShunJi; Zhang, ChengWu

    2012-01-01

    Background Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs) for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production. Results We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to >3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem. Conclusions Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock. PMID:22536352

  12. De novo transcriptomic analysis of an oleaginous microalga: pathway description and gene discovery for production of next-generation biofuels.

    Directory of Open Access Journals (Sweden)

    LingLin Wan

    Full Text Available BACKGROUND: Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production. RESULTS: We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to >3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem. CONCLUSIONS: Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock.

  13. A De Novo-Assembly Based Data Analysis Pipeline for Plant Obligate Parasite Metatranscriptomic Studies.

    Science.gov (United States)

    Guo, Li; Allen, Kelly S; Deiulio, Greg; Zhang, Yong; Madeiras, Angela M; Wick, Robert L; Ma, Li-Jun

    2016-01-01

    Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression analysis of a plant host and its obligate parasite simultaneously by next generation sequencing of mixed host and pathogen RNA (i.e., metatranscriptomics). We applied this pipeline to metatranscriptomic sequencing data of sweet basil (Ocimum basilicum) and its obligate downy mildew parasite Peronospora belbahrii, both lacking a sequenced genome. Even with a single data point, we were able to identify both candidate host defense genes and pathogen virulence genes that are highly expressed during infection. This demonstrates the power of this pipeline for identifying genes important in host-pathogen interactions without prior genomic information for either the plant host or the obligate biotrophic pathogen. The simplicity of this pipeline makes it accessible to researchers with limited computational skills and applicable to metatranscriptomic data analysis in a wide range of plant-obligate-parasite systems.

  14. A de-novo-assembly-based Data Analysis Pipeline for Plant Obligate Parasite Metatranscriptomic Studies

    Directory of Open Access Journals (Sweden)

    Li Guo

    2016-07-01

    Full Text Available Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression analysis of a plant host and its obligate parasite simultaneously by next generation sequencing of mixed host and pathogen RNA (i.e. metatranscriptomics. We applied this pipeline to metatranscriptomic sequencing data of sweet basil (Ocimum basilicum and its obligate downy mildew parasite Peronospora belbahrii, both lacking a sequenced genome. Even with a single data point, we were able to identify both candidate host defense genes and pathogen virulence genes that are highly expressed during infection. This demonstrates the power of this pipeline for identifying genes important in host-pathogen interactions without prior genomic information for either the plant host or the obligate biotrophic pathogen. The simplicity of this pipeline makes it accessible to researchers with limited computational skills and applicable to metatranscriptomic data analysis in a wide range of plant-obligate-parasite systems.

  15. A De Novo-Assembly Based Data Analysis Pipeline for Plant Obligate Parasite Metatranscriptomic Studies

    Science.gov (United States)

    Guo, Li; Allen, Kelly S.; Deiulio, Greg; Zhang, Yong; Madeiras, Angela M.; Wick, Robert L.; Ma, Li-Jun

    2016-01-01

    Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression analysis of a plant host and its obligate parasite simultaneously by next generation sequencing of mixed host and pathogen RNA (i.e., metatranscriptomics). We applied this pipeline to metatranscriptomic sequencing data of sweet basil (Ocimum basilicum) and its obligate downy mildew parasite Peronospora belbahrii, both lacking a sequenced genome. Even with a single data point, we were able to identify both candidate host defense genes and pathogen virulence genes that are highly expressed during infection. This demonstrates the power of this pipeline for identifying genes important in host–pathogen interactions without prior genomic information for either the plant host or the obligate biotrophic pathogen. The simplicity of this pipeline makes it accessible to researchers with limited computational skills and applicable to metatranscriptomic data analysis in a wide range of plant-obligate-parasite systems. PMID:27462318

  16. Serum Insulin-Like Growth Factor-1 in Patients with De Novo, Drug Naive Parkinson's Disease: A Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Dun-Hui Li

    Full Text Available Insulin-like growth factor-1 (IGF-1 is reported to be neuroprotective in the setting of Parkinson's disease (PD, and there is increasing interest in the possible association of serum IGF-1 levels with PD patients, but with conflicting results. Therefore, we conducted a meta-analysis to evaluate the association of serum IGF-1 levels in de novo, drug naïve PD patients compared with healthy controls.Pubmed, ISI Web of Science, OVID, EMBASE, and Cochrane library databases from 1966 to October 2014 were utilized to identify candidate studies using Medical Subjective Headings without language restriction. A random-effects model was chosen, with subgroup analysis and sensitivity analysis conducted to reveal underlying heterogeneity among the included studies.In this meta-analysis, we found that PD patients had higher serum IGF-1 levels compared with healthy controls (summary mean difference [MD] = 17.75, 95%CI = 6.01, 29.48. Subgroup analysis demonstrated that the source of heterogeneity was population differences within the total group. Sensitivity analysis showed that the combined MD was consistent at any time omitting any one study.The results of this meta-analysis demonstrate that serum IGF-1 levels were significantly higher in de novo, drug-naïve PD patients compared with healthy controls. Nevertheless, additional endeavors are required to further explore the association between serum IGF-1 levels and diagnosis, prognosis and early therapy for PD.

  17. De novo transcriptome analysis using 454 pyrosequencing of the Himalayan Mayapple, Podophyllum hexandrum.

    Science.gov (United States)

    Bhattacharyya, Dipto; Sinha, Ragini; Hazra, Saptarshi; Datta, Riddhi; Chattopadhyay, Sharmila

    2013-11-01

    The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic sequence data from the Indian Mayapple are essential for further understanding of the podophyllotoxin biosynthetic pathway. 454 pyrosequencing of a P. hexandrum cell culture normalized cDNA library generated 2,667,207 raw reads and 1,503,232 high quality reads, with an average read length of 138 bp. The denovo assembly was performed by Newbler using default and optimized parameters. The optimized parameter generated 40, 380 assembled sequences, comprising 12,940 contigs and 27,440 singlets which resulted in better assembly as compared to default parameters. BLASTX analysis resulted in the annotation of 40,380 contigs/singlet using a cut-off value of ≤ 1E-03. High similarity to Medicago truncatula using optimized parameters and to Populus trichocarpa using default parameters was noted. The Kyoto encyclopedia of genes and genomes (KEGG) analysis using KEGG Automatic Annotation Server (KAAS) combined with domain analysis of the assembled transcripts revealed putative members of secondary metabolism pathways that may be involved in podophyllotoxin biosynthesis. A proposed schematic pathway for phenylpropanoids and podophyllotoxin biosynthesis was generated. Expression profiling was carried out based on fragments per kilobase of exon per million fragments (FPKM). 1036 simple sequence repeats were predicted in the P. hexandrum sequences. Sixty-nine transcripts were mapped to 99 mature and precursor microRNAs from the plant microRNA database. Around 961 transcripts containing transcription factor domains were noted. High performance liquid chromatography analysis showed the peak accumulation of podophyllotoxin in 12-day cell suspension cultures. A comparative qRT-PCR analysis of phenylpropanoid pathway genes identified in the present data was performed to analyze

  18. Sequence Matching Analysis for Curriculum Development

    National Research Council Canada - National Science Library

    Liem Yenny Bendatu; Bernardo Nugroho Yahya

    2015-01-01

    .... This study attempts to develop a sequence matching analysis. Considering conformance checking as the basis of this approach, this proposed approach utilizes the current control flow technique in process mining domain...

  19. Comparative analysis of four essential Gracilariaceae species in China based on whole transcriptomic sequencing

    Institute of Scientific and Technical Information of China (English)

    XU Jiayue; WU Shuangxiu; YU Jun; SUN Jing; YIN Jinlong; WANG Liang; WANG Xumin; LIU Tao; CHI Shan; LIU Cui; REN Lufeng

    2014-01-01

    Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilari-opsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous spe-cies which are important resources for the production of special proteins, phycobilisomes, special carbo-hydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lema-neiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.

  20. De novo quence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue-green alga Aphanizomenon flos-aquae

    DEFF Research Database (Denmark)

    Rinalducci, Sara; Roepstorff, Peter; Zolla, Lello

    2009-01-01

    In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos-aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N-terminal sulfonation of tryptic peptides by 4-sulfophenyl...

  1. De novo assembly and characterization of farmed blue fox (Alopex lagopus) global transcriptome using Illumina paired-end sequencing.

    Science.gov (United States)

    Guo, P C; Yan, S Q; Si, S; Bai, C Y; Zhao, Y; Zhang, Y; Yao, J Y; Li, Y M

    2016-03-28

    The blue fox (Alopex lagopus), a coat-color variant of the Arctic fox, is a domesticated fur-bearing mammal. In the present study, transcriptome data generated from a pool of nine different tissues were obtained with Illumina HiSeq2500 paired-end sequencing technology. After filtering from raw reads, 32,358,290 clean reads were assembled into 161,269 transcripts and 97,252 unigenes by the Trinity fragment assembly software. Of the assembled unigenes, 37,967 were annotated in the National Center for Biotechnology Information (NCBI) Non-Redundant (NR) protein database and 26,264 in the Swiss-Prot database. Among the annotated unigenes, 24,839 and 24,267 were assigned using the Gene Ontology (GO) and euKaryotic Orthologous Groups (KOG) databases, respectively. Altogether, 17,057 unigenes were mapped onto 227 pathways using the Kyoto Encyclopedia of Genes and Genomes database. In addition, 6394 simple sequence repeats were identified by examining 12,965 unigenes (>1 kb), which could contribute to the development of molecular markers. This study generated transcriptome data for the blue fox that will promote further progress in expression profiling studies, and provide a good annotation basis for genomic studies.

  2. A De Novo Whole GCK Gene Deletion Not Detected by Gene Sequencing, in a Boy with Phenotypic GCK Insufficiency

    Directory of Open Access Journals (Sweden)

    N. H. Birkebæk

    2011-01-01

    Full Text Available We report on a boy with diabetes mellitus and a phenotype indicating glucokinase (GCK insufficiency, but a normal GCK gene examination applying direct gene sequencing. The boy was referred for diabetes mellitus at 7.5 years old. His father, grandfather and great grandfather suffered type 2 DM. Several blood glucose profiles showed (BG of 6.5–10 mmol/L L. After three years on neutral insulin Hagedorn (NPH in a dose of 0.3 IU/kg/day haemoglobin A1c (HbA1c was 6.8%. Treatment was changed to sulphonylurea 750 mg a day, and after 4 years HbA1c was 7%. At that time a multiplex ligation-dependent amplification gene dosage assay (MLPA was done, revealing a whole GCK gene deletion. Medical treatment was ceased, and after one year HbA1c was 6.8%. This case underscores the importance of a MLPA examination if the phenotype of a patient is strongly indicative of GCK insufficiency and no mutation is identified using direct sequencing.

  3. Scale-PC shielding analysis sequences

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, S.M.

    1996-05-01

    The SCALE computational system is a modular code system for analyses of nuclear fuel facility and package designs. With the release of SCALE-PC Version 4.3, the radiation shielding analysis community now has the capability to execute the SCALE shielding analysis sequences contained in the control modules SAS1, SAS2, SAS3, and SAS4 on a MS- DOS personal computer (PC). In addition, SCALE-PC includes two new sequences, QADS and ORIGEN-ARP. The capabilities of each sequence are presented, along with example applications.

  4. De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

    Directory of Open Access Journals (Sweden)

    Wu Xiwei

    2012-03-01

    Full Text Available Abstract Background MicroRNAs (miRNAs have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC, and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome. Methods The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers. Results More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients. Conclusions Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

  5. Multiplexed next-generation sequencing and de novo assembly to obtain near full-length HIV-1 genome from plasma virus.

    Science.gov (United States)

    Aralaguppe, Shambhu G; Siddik, Abu Bakar; Manickam, Ashokkumar; Ambikan, Anoop T; Kumar, Milner M; Fernandes, Sunjay Jude; Amogne, Wondwossen; Bangaruswamy, Dhinoth K; Hanna, Luke Elizabeth; Sonnerborg, Anders; Neogi, Ujjwal

    2016-10-01

    Analysing the HIV-1 near full-length genome (HIV-NFLG) facilitates new understanding into the diversity of virus population dynamics at individual or population level. In this study we developed a simple but high-throughput next generation sequencing (NGS) protocol for HIV-NFLG using clinical specimens and validated the method against an external quality control (EQC) panel. Clinical specimens (n=105) were obtained from three cohorts from two highly conserved HIV-1C epidemics (India and Ethiopia) and one diverse epidemic (Sweden). Additionally an EQC panel (n=10) was used to validate the protocol. HIV-NFLG was performed amplifying the HIV-genome (Gag-to-nef) in two fragments. NGS was performed using the Illumina HiSeq2500 after multiplexing 24 samples, followed by de novo assembly in Iterative Virus Assembler or VICUNA. Subtyping was carried out using several bioinformatics tools. Amplification of HIV-NFLG has 90% (95/105) success-rate in clinical specimens. NGS was successful in all clinical specimens (n=45) and EQA samples (n=10) attempted. The mean error for mutations for the EQC panel viruses were <1%. Subtyping identified two as A1C recombinant. Our results demonstrate the feasibility of a simple NGS-based HIV-NFLG that can potentially be used in the molecular surveillance for effective identification of subtypes and transmission clusters for operational public health intervention.

  6. De novo assembly and analysis of Cassia obtusifolia seed transcriptome to identify genes involved in the biosynthesis of active metabolites.

    Science.gov (United States)

    Liu, Zubi; Song, Tao; Zhu, Qiankun; Wang, Wanjun; Zhou, Jiayu; Liao, Hai

    2014-01-01

    A cDNA library generated from seeds of Cassia obtusifolia was sequenced using Illumina/Solexa platform. More than 12,968,231 high quality reads were generated, and have been deposited in NCBI SRA (SRR 1012912). A total of 40,102 unigenes (>200 bp) were obtained with an average sequence length of 681 bp by de novo assembly. About 34,089 (85%) unique sequences were annotated and 8694 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Among them, 131 unigenes, which are involved in the biosynthesis and (or) regulation of anthraquinone, carotenoid, flavonoid, and lipid, the 4 best known active metabolites, were identified from cDNA library. In addition, three lipid transfer proteins were obtained, which may contribute to the lipid molecules transporting between biological membranes. Meanwhile, 30 cytochrome P450, 12 SAM-dependent methyltransferases, and 12 UDP-glucosyltransferase unigenes were identified, which could also be responsible for the biosynthesis of active metabolites.

  7. De novo transcriptome analysis of the red seaweed Gracilaria chilensis and identification of linkers associated with phycobilisomes.

    Science.gov (United States)

    Vorphal, María Alejandra; Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Dagnino-Leone, Jorge; Vásquez, José Aleikar; Martínez-Oyanedel, José; Bunster, Marta

    2017-02-01

    This work reports the results of the Illumina RNA-Seq of a wild population of female haploid plants of Gracilaria chilensis (Bird et al., 1986) (Rhodophyta, Gigartinalis). Most transcripts were de novo assembled in 12,331 contigs with an average length of 1756bp, showing that 96.64% of the sequences were annotated with known proteins. In particular, the identification of linker proteins of phycobilisomes (PBS) is reported. Linker proteins have primary been identified in cyanobacteria but the information available about them in eukaryotic red alga is not complete, and this is the first report in G. chilensis. This resource will also provide the basis for the study of metabolic pathways related to polysaccharide production.

  8. De novo transcriptome sequence and identification of major bast-related genes involved in cellulose biosynthesis in jute (Corchorus capsularis L.).

    Science.gov (United States)

    Zhang, Liwu; Ming, Ray; Zhang, Jisen; Tao, Aifen; Fang, Pingping; Qi, Jianmin

    2015-12-15

    Jute fiber, extracted from stem bast, is called golden fiber. It is essential for fiber improvement to discover the genes associated with jute development at the vegetative growth stage. However, only 858 EST sequences of jute were deposited in the GenBank database. Obviously, the public available data is far from sufficient to understand the molecular mechanism of the fiber biosynthesis. It is imperative to conduct transcriptomic sequence for jute, which can be used for the discovery of a number of new genes, especially genes involved in cellulose biosynthesis. A total of 79,754,600 clean reads (7.98 Gb) were generated using Illumina paired-end sequencing. De novo assembly yielded 48,914 unigenes with an average length of 903 bp. By sequence similarity searching for known proteins, 27,962 (57.16 %) unigenes were annotated for their function. Out of these annotated unigenes, 21,856 and 11,190 unigenes were assigned to gene ontology (GO) and euKaryotic Ortholog Groups (KOG), respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 14,216 unigenes were mapped to 268 KEGG pathways. Moreover, 5 Susy, 3 UGPase, 9 CesA, 18 CSL, 2 Kor (Korrigan), and 12 Cobra unigenes involving in cellulose biosynthesis were identified. Among these unigenes, the unigenes of comp11264_c0 (SuSy), comp24568_c0 (UGPase), comp11363_c0 (CesA), comp11363_c1 (CesA), comp24217_c0 (CesA), and comp23531_c0 (CesA), displayed relatively high expression level in stem bast using FPKM and RT-qPCR, indicating that they may have potential value of dissecting mechanism on cellulose biosynthesis in jute. In addition, a total of 12,518 putative gene-associate SNPs were called from these assembled uingenes. We characterized the transcriptome of jute, discovered a broad survey of unigenes associated with vegetative growth and development, developed large-scale SNPs, and analyzed the expression patterns of genes involved in cellulose biosynthesis for bast

  9. SNP detection from de novo transcriptome sequencing in the bivalve Macoma balthica: marker development for evolutionary studies.

    Directory of Open Access Journals (Sweden)

    Eric Pante

    Full Text Available Hybrid zones are noteworthy systems for the study of environmental adaptation to fast-changing environments, as they constitute reservoirs of polymorphism and are key to the maintenance of biodiversity. They can move in relation to climate fluctuations, as temperature can affect both selection and migration, or remain trapped by environmental and physical barriers. There is therefore a very strong incentive to study the dynamics of hybrid zones subjected to climate variations. The infaunal bivalve Macoma balthica emerges as a noteworthy model species, as divergent lineages hybridize, and its native NE Atlantic range is currently contracting to the North. To investigate the dynamics and functioning of hybrid zones in M. balthica, we developed new molecular markers by sequencing the collective transcriptome of 30 individuals. Ten individuals were pooled for each of the three populations sampled at the margins of two hybrid zones. A single 454 run generated 277 Mb from which 17K SNPs were detected. SNP density averaged 1 polymorphic site every 14 to 19 bases, for mitochondrial and nuclear loci, respectively. An [Formula: see text] scan detected high genetic divergence among several hundred SNPs, some of them involved in energetic metabolism, cellular respiration and physiological stress. The high population differentiation, recorded for nuclear-encoded ATP synthase and NADH dehydrogenase as well as most mitochondrial loci, suggests cytonuclear genetic incompatibilities. Results from this study will help pave the way to a high-resolution study of hybrid zone dynamics in M. balthica, and the relative importance of endogenous and exogenous barriers to gene flow in this system.

  10. Sequencing and Analysis of Neanderthal Genomic DNA

    OpenAIRE

    Noonan, James P.; Coop, Graham; Kudaravalli, Sridhar; Smith, Doug; Krause, Johannes; Alessi, Joe; Chen, Feng; Platt, Darren; Paabo, Svante; Pritchard, Jonathan K; Rubin, Edward M.

    2006-01-01

    Our knowledge of Neanderthals is based on a limited number of remains and artifacts from which we must make inferences about their biology, behavior, and relationship to ourselves. Here, we describe the characterization of these extinct hominids from a new perspective, based on the development of a Neanderthal metagenomic library and its high-throughput sequencing and analysis. Several lines of evidence indicate that the 65,250 base pairs of hominid sequence so far identified in the library a...

  11. Digital gene expression analysis based on integrated de novo transcriptome assembly of sweet potato [Ipomoea batatas (L. Lam].

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    Xiang Tao

    Full Text Available BACKGROUND: Sweet potato (Ipomoea batatas L. [Lam.] ranks among the top six most important food crops in the world. It is widely grown throughout the world with high and stable yield, strong adaptability, rich nutrient content, and multiple uses. However, little is known about the molecular biology of this important non-model organism due to lack of genomic resources. Hence, studies based on high-throughput sequencing technologies are needed to get a comprehensive and integrated genomic resource and better understanding of gene expression patterns in different tissues and at various developmental stages. METHODOLOGY/PRINCIPAL FINDINGS: Illumina paired-end (PE RNA-Sequencing was performed, and generated 48.7 million of 75 bp PE reads. These reads were de novo assembled into 128,052 transcripts (≥ 100 bp, which correspond to 41.1 million base pairs, by using a combined assembly strategy. Transcripts were annotated by Blast2GO and 51,763 transcripts got BLASTX hits, in which 39,677 transcripts have GO terms and 14,117 have ECs that are associated with 147 KEGG pathways. Furthermore, transcriptome differences of seven tissues were analyzed by using Illumina digital gene expression (DGE tag profiling and numerous differentially and specifically expressed transcripts were identified. Moreover, the expression characteristics of genes involved in viral genomes, starch metabolism and potential stress tolerance and insect resistance were also identified. CONCLUSIONS/SIGNIFICANCE: The combined de novo transcriptome assembly strategy can be applied to other organisms whose reference genomes are not available. The data provided here represent the most comprehensive and integrated genomic resources for cloning and identifying genes of interest in sweet potato. Characterization of sweet potato transcriptome provides an effective tool for better understanding the molecular mechanisms of cellular processes including development of leaves and storage roots

  12. RSAT 2015: Regulatory Sequence Analysis Tools.

    Science.gov (United States)

    Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

    2015-07-01

    RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/.

  13. Analysis of the NovoTwist pen needle in comparison with conventional screw-thread needles.

    Science.gov (United States)

    Aye, Tandy

    2011-11-01

    Administration of insulin via a pen device may be advantageous over a vial and syringe system. Hofman and colleagues introduce a new insulin pen needle, the NovoTwist, to simplify injections to a small group of children and adolescents. Their overall preferences and evaluation of the handling of the needle are reported in the study. This new needle has the potential to ease administration of insulin via a pen device that may increase both the use of a pen device and adherence to insulin therapy.

  14. Sequence analysis by iterated maps, a review.

    Science.gov (United States)

    Almeida, Jonas S

    2014-05-01

    Among alignment-free methods, Iterated Maps (IMs) are on a particular extreme: they are also scale free (order free). The use of IMs for sequence analysis is also distinct from other alignment-free methodologies in being rooted in statistical mechanics instead of computational linguistics. Both of these roots go back over two decades to the use of fractal geometry in the characterization of phase-space representations. The time series analysis origin of the field is betrayed by the title of the manuscript that started this alignment-free subdomain in 1990, 'Chaos Game Representation'. The clash between the analysis of sequences as continuous series and the better established use of Markovian approaches to discrete series was almost immediate, with a defining critique published in same journal 2 years later. The rest of that decade would go by before the scale-free nature of the IM space was uncovered. The ensuing decade saw this scalability generalized for non-genomic alphabets as well as an interest in its use for graphic representation of biological sequences. Finally, in the past couple of years, in step with the emergence of BigData and MapReduce as a new computational paradigm, there is a surprising third act in the IM story. Multiple reports have described gains in computational efficiency of multiple orders of magnitude over more conventional sequence analysis methodologies. The stage appears to be now set for a recasting of IMs with a central role in processing nextgen sequencing results.

  15. De Novo Transcriptome Analysis of Allium cepa L. (Onion Bulb to Identify Allergens and Epitopes.

    Directory of Open Access Journals (Sweden)

    Hemalatha Rajkumar

    Full Text Available Allium cepa (onion is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb. Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins.

  16. De Novo Transcriptome Analysis of Allium cepa L. (Onion) Bulb to Identify Allergens and Epitopes.

    Science.gov (United States)

    Rajkumar, Hemalatha; Ramagoni, Ramesh Kumar; Anchoju, Vijayendra Chary; Vankudavath, Raju Naik; Syed, Arshi Uz Zaman

    2015-01-01

    Allium cepa (onion) is a diploid plant with one of the largest nuclear genomes among all diploids. Onion is an example of an under-researched crop which has a complex heterozygous genome. There are no allergenic proteins and genomic data available for onions. This study was conducted to establish a transcriptome catalogue of onion bulb that will enable us to study onion related genes involved in medicinal use and allergies. Transcriptome dataset generated from onion bulb using the Illumina HiSeq 2000 technology showed a total of 99,074,309 high quality raw reads (~20 Gb). Based on sequence homology onion genes were categorized into 49 different functional groups. Most of the genes however, were classified under 'unknown' in all three gene ontology categories. Of the categorized genes, 61.2% showed metabolic functions followed by cellular components such as binding, cellular processes; catalytic activity and cell part. With BLASTx top hit analysis, a total of 2,511 homologous allergenic sequences were found, which had 37-100% similarity with 46 different types of allergens existing in the database. From the 46 contigs or allergens, 521 B-cell linear epitopes were identified using BepiPred linear epitope prediction tool. This is the first comprehensive insight into the transcriptome of onion bulb tissue using the NGS technology, which can be used to map IgE epitopes and prediction of structures and functions of various proteins.

  17. Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

    Directory of Open Access Journals (Sweden)

    Roberto Pérez-Torrado

    Full Text Available In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

  18. De novo Assembly and Analysis of the Chilean Pencil Catfish Trichomycterus areolatus Transcriptome

    Science.gov (United States)

    Schulze, Thomas T.; Ali, Jonathan M.; Bartlett, Maggie L.; McFarland, Madalyn M.; Clement, Emalie J.; Won, Harim I.; Sanford, Austin G.; Monzingo, Elyssa B.; Martens, Matthew C.; Hemsley, Ryan M.; Kumar, Sidharta; Gouin, Nicolas; Kolok, Alan S.; Davis, Paul H.

    2016-01-01

    Trichomycterus areolatus is an endemic species of pencil catfish that inhabits the riffles and rapids of many freshwater ecosystems of Chile. Despite its unique adaptation to Chile's high gradient watersheds and therefore potential application in the investigation of ecosystem integrity and environmental contamination, relatively little is known regarding the molecular biology of this environmental sentinel. Here, we detail the assembly of the Trichomycterus areolatus transcriptome, a molecular resource for the study of this organism and its molecular response to the environment. RNA-Seq reads were obtained by next-generation sequencing with an Illumina® platform and processed using PRINSEQ. The transcriptome assembly was performed using TRINITY assembler. Transcriptome validation was performed by functional characterization with KOG, KEGG, and GO analyses. Additionally, differential expression analysis highlights sex-specific expression patterns, and a list of endocrine and oxidative stress related transcripts are included. PMID:27672404

  19. Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation

    DEFF Research Database (Denmark)

    Czaban, Adrian; Sharma, Sapna; Byrne, Stephen

    2015-01-01

    -Festuca complex show very diverse phenotypes, including for many agronomically important traits. Analysis of sequenced transcriptomes of these non-model species may shed light on the molecular mechanisms underlying this phenotypic diversity. Results We have generated de novo transcriptome assemblies for four...... clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity...

  20. De novo transcriptome analysis of wing development-related signaling pathways in Locusta migratoria manilensis and Ostrinia furnacalis (Guenee.

    Directory of Open Access Journals (Sweden)

    Suning Liu

    Full Text Available BACKGROUND: Orthopteran migratory locust, Locusta migratoria, and lepidopteran Asian corn borer, Ostrinia furnacalis, are two types of insects undergoing incomplete and complete metamorphosis, respectively. Identification of candidate genes regulating wing development in these two insects would provide insights into the further study about the molecular mechanisms controlling metamorphosis development. We have sequenced the transcriptome of O. furnacalis larvae previously. Here we sequenced and characterized the transcriptome of L. migratoria wing discs with special emphasis on wing development-related signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: Illumina Hiseq2000 was used to sequence 8.38 Gb of the transcriptome from dissected nymphal wing discs. De novo assembly generated 91,907 unigenes with mean length of 610 nt. All unigenes were searched against five databases including Nt, Nr, Swiss-Prot, COG, and KEGG for annotations using blastn or blastx algorithm with an cut-off E-value of 10-5. A total of 23,359 (25.4% unigenes have homologs within at least one database. Based on sequence similarity to homologs known to regulate Drosophila melanogaster wing development, we identified 50 and 46 potential wing development-related unigenes from L. migratoria and O. furnacalis transcriptome, respectively. The identified unigenes encode putative orthologs for nearly all components of the Hedgehog (Hh, Decapentaplegic (Dpp, Notch (N, and Wingless (Wg signaling pathways, which are essential for growth and pattern formation during wing development. We investigated the expression profiles of the component genes involved in these signaling pathways in forewings and hind wings of L. migratoria and O. furnacalis. The results revealed the tested genes had different expression patterns in two insects. CONCLUSIONS/SIGNIFICANCE: This study provides the comprehensive sequence resource of the wing development-related signaling pathways of L. migratoria. The

  1. Cladistic analysis of anuran POMC sequences.

    Science.gov (United States)

    Alrubaian, Jasem; Danielson, Phillip; Walker, David; Dores, Robert M

    2002-03-01

    Procedures for performing cladistic analyses can provide powerful tools for understanding the evolution of neuropeptide and polypeptide hormone coding genes. These analyses can be done on either amino acid data sets or nucleotide data sets and can utilize several different algorithms that are dependent on distinct sets of operating assumptions and constraints. In some cases, the results of these analyses can be used to gauge phylogenetic relationships between taxa. Selecting the proper cladistic analysis strategy is dependent on the taxonomic level of analysis and the rate of evolution within the orthologous genes being evaluated. For example, previous studies have shown that the amino acid sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, can be used to resolve phylogenetic relationships at the class and order level. This study tested the hypothesis that POMC sequences could be used to resolve phylogenetic relationships at the family taxonomic level. Cladistic analyses were performed on amphibian POMC sequences characterized from the marine toad, Bufo marinus (family Bufonidae; this study), the spadefoot toad, Spea multiplicatus (family Pelobatidae), the African clawed frog, Xenopus laevis (family Pipidae) and the laughing frog, Rana ridibunda (family Ranidae). In these analyses the sequence of Australian lungfish POMC was used as the outgroup. The analyses were done at the amino acid level using the maximum parsimony algorithm and at the nucleotide level using the maximum likelihood algorithm. For the anuran POMC genes, analysis at the nucleotide level using the maximum likelihood algorithm generated a cladogram with higher bootstrap values than the maximum parsimony analysis of the POMC amino acid data set. For anuran POMC sequences, analysis of nucleotide sequences using the maximum likelihood algorithm would appear to be the preferred strategy for resolving phylogenetic relationships at the family taxonomic

  2. A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery.

    Science.gov (United States)

    Thanh, Nguyen Minh; Jung, Hyungtaek; Lyons, Russell E; Chand, Vincent; Tuan, Nguyen Viet; Thu, Vo Thi Minh; Mather, Peter

    2014-06-01

    The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9ppt which showed best growth performance. Total sequence data generated was 467.8Mbp, consisting of 4,116,424 reads with an average length of 112bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

  3. De Novo characterization of the banana root transcriptome and analysis of gene expression under Fusarium oxysporum f. sp. Cubense tropical race 4 infection

    Directory of Open Access Journals (Sweden)

    Wang Zhuo

    2012-11-01

    Full Text Available Abstract Background Bananas and plantains (Musa spp. are among the most important crops in the world due to their nutritional and export value. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc, which cannot be effectively prevented or controlled. Since there is very little known about the molecular mechanism of Foc infections; therefore, we aimed to investigate the transcriptional changes induced by Foc in banana roots. Results We generated a cDNA library from total RNA isolated from banana roots infected with Foc Tropical Race 4 (Foc TR 4 at days 0, 2, 4, and 6. We generated over 26 million high-quality reads from the cDNA library using deep sequencing and assembled 25,158 distinct gene sequences by de novo assembly and gap-filling. The average distinct gene sequence length was 1,439 base pairs. A total of 21,622 (85.94% unique sequences were annotated and 11,611 were assigned to specific metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes database. We used digital gene expression (DGE profiling to investigate the transcriptional changes in the banana root upon Foc TR4 infection. The expression of genes in the Phenylalanine metabolism, phenylpropanoid biosynthesis and alpha-linolenic acid metabolism pathways was affected by Foc TR4 infection. Conclusion The combination of RNA-Seq and DGE analysis provides a powerful method for analyzing the banana root transcriptome and investigating the transcriptional changes during the response of banana genes to Foc TR4 infection. The assembled banana transcriptome provides an important resource for future investigations about the banana crop as well as the diseases that plague this valuable staple food.

  4. De novo assembly and characterization of the transcriptome of the pancreatic fluke Eurytrema pancreaticum (trematoda: Dicrocoeliidae) using Illumina paired-end sequencing.

    Science.gov (United States)

    Liu, Guo-Hua; Xu, Min-Jun; Song, Hui-Qun; Wang, Chun-Ren; Zhu, Xing-Quan

    2016-01-15

    Eurytrema pancreaticum is one of the most common trematodes living in the pancreatic and bile ducts of ruminants and also occasionally infects humans, causing eurytremiasis. In spite of its economic and medical importance, very little is known about the genomic resources of this parasite. Herein, we performed de novo sequencing, assembly and characterization of the transcriptome of adult E. pancreaticum. Approximately 36.4 million high-quality clean reads were obtained, and the length of the transcript contigs ranged from 66 to 19,968 nt with mean length of 479 nt and N50 length of 1094 nt, and then 23,573 unigenes were assembled. Of these unigenes, 15,353 (65.1%) were annotated by blast searches against the NCBI non-redundant protein database. Among these, 15,267 (64.8%), 2732 (11.6%) and 10,354 (43.9%) of the unigenes had significant similarity with proteins in the NR, NT and Swiss-Prot databases, respectively. 5510 (23.4%) and 4567 (19.4%) unigenes were assigned to GO and COG, respectively. 8886 (37.7%) unigenes were identified and mapped onto 254 pathways in the KEGG Pathway database. Furthermore, we found that 105 (1.18%) unigenes were related to pancreatic secretion and 61 (0.7%) to pancreatic cancer. The present study represents the first transcriptome of any members of the family Dicrocoeliidae, which has little genomic information available in the public databases. The novel transcriptome of E. pancreaticum should provide a useful resource for designing new strategies against pancreatic flukes and other trematodes of human and animal health significance. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Information theory applications for biological sequence analysis.

    Science.gov (United States)

    Vinga, Susana

    2014-05-01

    Information theory (IT) addresses the analysis of communication systems and has been widely applied in molecular biology. In particular, alignment-free sequence analysis and comparison greatly benefited from concepts derived from IT, such as entropy and mutual information. This review covers several aspects of IT applications, ranging from genome global analysis and comparison, including block-entropy estimation and resolution-free metrics based on iterative maps, to local analysis, comprising the classification of motifs, prediction of transcription factor binding sites and sequence characterization based on linguistic complexity and entropic profiles. IT has also been applied to high-level correlations that combine DNA, RNA or protein features with sequence-independent properties, such as gene mapping and phenotype analysis, and has also provided models based on communication systems theory to describe information transmission channels at the cell level and also during evolutionary processes. While not exhaustive, this review attempts to categorize existing methods and to indicate their relation with broader transversal topics such as genomic signatures, data compression and complexity, time series analysis and phylogenetic classification, providing a resource for future developments in this promising area.

  6. Digital image sequence processing, compression, and analysis

    CERN Document Server

    Reed, Todd R

    2004-01-01

    IntroductionTodd R. ReedCONTENT-BASED IMAGE SEQUENCE REPRESENTATIONPedro M. Q. Aguiar, Radu S. Jasinschi, José M. F. Moura, andCharnchai PluempitiwiriyawejTHE COMPUTATION OF MOTIONChristoph Stiller, Sören Kammel, Jan Horn, and Thao DangMOTION ANALYSIS AND DISPLACEMENT ESTIMATION IN THE FREQUENCY DOMAINLuca Lucchese and Guido Maria CortelazzoQUALITY OF SERVICE ASSESSMENT IN NEW GENERATION WIRELESS VIDEO COMMUNICATIONSGaetano GiuntaERROR CONCEALMENT IN DIGITAL VIDEOFrancesco G.B. De NataleIMAGE SEQUENCE RESTORATION: A WIDER PERSPECTIVEAnil KokaramVIDEO SUMMARIZATIONCuneyt M. Taskiran and Edward

  7. ANGSD: Analysis of Next Generation Sequencing Data.

    Science.gov (United States)

    Korneliussen, Thorfinn Sand; Albrechtsen, Anders; Nielsen, Rasmus

    2014-11-25

    High-throughput DNA sequencing technologies are generating vast amounts of data. Fast, flexible and memory efficient implementations are needed in order to facilitate analyses of thousands of samples simultaneously. We present a multithreaded program suite called ANGSD. This program can calculate various summary statistics, and perform association mapping and population genetic analyses utilizing the full information in next generation sequencing data by working directly on the raw sequencing data or by using genotype likelihoods. The open source c/c++ program ANGSD is available at http://www.popgen.dk/angsd . The program is tested and validated on GNU/Linux systems. The program facilitates multiple input formats including BAM and imputed beagle genotype probability files. The program allow the user to choose between combinations of existing methods and can perform analysis that is not implemented elsewhere.

  8. OTU analysis using metagenomic shotgun sequencing data.

    Directory of Open Access Journals (Sweden)

    Xiaolin Hao

    Full Text Available Because of technological limitations, the primer and amplification biases in targeted sequencing of 16S rRNA genes have veiled the true microbial diversity underlying environmental samples. However, the protocol of metagenomic shotgun sequencing provides 16S rRNA gene fragment data with natural immunity against the biases raised during priming and thus the potential of uncovering the true structure of microbial community by giving more accurate predictions of operational taxonomic units (OTUs. Nonetheless, the lack of statistically rigorous comparison between 16S rRNA gene fragments and other data types makes it difficult to interpret previously reported results using 16S rRNA gene fragments. Therefore, in the present work, we established a standard analysis pipeline that would help confirm if the differences in the data are true or are just due to potential technical bias. This pipeline is built by using simulated data to find optimal mapping and OTU prediction methods. The comparison between simulated datasets revealed a relationship between 16S rRNA gene fragments and full-length 16S rRNA sequences that a 16S rRNA gene fragment having a length >150 bp provides the same accuracy as a full-length 16S rRNA sequence using our proposed pipeline, which could serve as a good starting point for experimental design and making the comparison between 16S rRNA gene fragment-based and targeted 16S rRNA sequencing-based surveys possible.

  9. Comparative transcriptome sequencing and de novo analysis of Vaccinium corymbosum during fruit and color development

    OpenAIRE

    Li, Lingli; Zhang, Hehua; Liu, Zhongshuai; Cui, Xiaoyue; Zhang, Tong; Li, Yanfang; Zhang, Lingyun

    2016-01-01

    Background Blueberry is an economically important fruit crop in Ericaceae family. The substantial quantities of flavonoids in blueberry have been implicated in a broad range of health benefits. However, the information regarding fruit development and flavonoid metabolites based on the transcriptome level is still limited. In the present study, the transcriptome and gene expression profiling over berry development, especially during color development were initiated. Results A total of approxim...

  10. Sequence Matching Analysis for Curriculum Development

    Directory of Open Access Journals (Sweden)

    Liem Yenny Bendatu

    2015-06-01

    Full Text Available Many organizations apply information technologies to support their business processes. Using the information technologies, the actual events are recorded and utilized to conform with predefined model. Conformance checking is an approach to measure the fitness and appropriateness between process model and actual events. However, when there are multiple events with the same timestamp, the traditional approach unfit to result such measures. This study attempts to develop a sequence matching analysis. Considering conformance checking as the basis of this approach, this proposed approach utilizes the current control flow technique in process mining domain. A case study in the field of educational process has been conducted. This study also proposes a curriculum analysis framework to test the proposed approach. By considering the learning sequence of students, it results some measurements for curriculum development. Finally, the result of the proposed approach has been verified by relevant instructors for further development.

  11. RIKEN Integrated Sequence Analysis (RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer

    OpenAIRE

    Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Nagaoka, Sumiharu; Sasaki, Nobuya; Carninci, Piero; Konno, Hideaki; AKIYAMA, Junichi; Nishi, Katsuo; Kitsunai, Tokuji; Tashiro, Hideo; Itoh, Mari; Sumi, Noriko; Ishii, Yoshiyuki; Nakamura, Shin

    2000-01-01

    The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto seque...

  12. De novo assembly and characterization of global transcriptome of coconut palm (Cocos nucifera L.) embryogenic calli using Illumina paired-end sequencing.

    Science.gov (United States)

    Rajesh, M K; Fayas, T P; Naganeeswaran, S; Rachana, K E; Bhavyashree, U; Sajini, K K; Karun, Anitha

    2016-05-01

    Production and supply of quality planting material is significant to coconut cultivation but is one of the major constraints in coconut productivity. Rapid multiplication of coconut through in vitro techniques, therefore, is of paramount importance. Although somatic embryogenesis in coconut is a promising technique that will allow for the mass production of high quality palms, coconut is highly recalcitrant to in vitro culture. In order to overcome the bottlenecks in coconut somatic embryogenesis and to develop a repeatable protocol, it is imperative to understand, identify, and characterize molecular events involved in coconut somatic embryogenesis pathway. Transcriptome analysis (RNA-Seq) of coconut embryogenic calli, derived from plumular explants of West Coast Tall cultivar, was undertaken on an Illumina HiSeq 2000 platform. After de novo transcriptome assembly and functional annotation, we have obtained 40,367 transcripts which showed significant BLASTx matches with similarity greater than 40 % and E value of ≤10(-5). Fourteen genes known to be involved in somatic embryogenesis were identified. Quantitative real-time PCR (qRT-PCR) analyses of these 14 genes were carried in six developmental stages. The result showed that CLV was upregulated in the initial stage of callogenesis. Transcripts GLP, GST, PKL, WUS, and WRKY were expressed more in somatic embryo stage. The expression of SERK, MAPK, AP2, SAUR, ECP, AGP, LEA, and ANT were higher in the embryogenic callus stage compared to initial culture and somatic embryo stages. This study provides the first insights into the gene expression patterns during somatic embryogenesis in coconut.

  13. Genome sequencing and analysis of a granulovirus isolated from the Asiatic rice leafroller, Cnaphalocrocis medinalis.

    Science.gov (United States)

    Zhang, Shan; Zhu, Zheng; Sun, Shifeng; Chen, Qijin; Deng, Fei; Yang, Kai

    2015-12-01

    The complete genome of Cnaphalocrocis medinalis granulovirus (CnmeGV) from a serious migratory rice pest, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), was sequenced using the Roche 454 Genome Sequencer FLX system (GS FLX) with shotgun strategy and assembled by Roche GS De Novo assembler software. Its circular double-stranded genome is 111,246 bp in size with a high A+T content of 64.8% and codes for 118 putative open reading frames (ORFs). It contains 37 conserved baculovirus core ORFs, 13 unique ORFs, 26 ORFs that were found in all Lepidoptera baculoviruses and 42 common ORFs. The analysis of nucleotide sequence repeats revealed that the CnmeGV genome differs from the rest of sequenced GVs by a 23 kb and a 17kb gene block inversions, and does not contain any typical homologous region (hr) except for a region of non-hr-like sequence. Chitinase and cathepsin genes, which are reported to have major roles in the liquefaction of the hosts, were not found in the CnmeGV genome, which explains why CnmeGV infected insects do not show the phenotype of typical liquefaction. Phylogenetic analysis, based on the 37 core baculovirus genes, indicates that CnmeGV is closely related to Adoxophyes orana granulovirus. The genome analysis would contribute to the functional research of CnmeGV, and would benefit to the utilization of CnmeGV as pest control reagent for rice production.

  14. Genome sequencing and analysis of a granulovirus isolated from the Asiatic rice leafroller, Cnaphalocrocis medinalis

    Institute of Scientific and Technical Information of China (English)

    Shan Zhang; Zheng Zhu; Shifeng Sun; Qijin Chen; Fei Deng; Kai Yang

    2015-01-01

    The complete genome of Cnaphalocrocis medinalis granulovirus(CnmeGV) from a serious migratory rice pest, Cnaphalocrocis medinalis(Lepidoptera: Pyralidae), was sequenced using the Roche 454 Genome Sequencer FLX system(GS FLX) with shotgun strategy and assembled by Roche GS De Novo assembler software. Its circular double-stranded genome is 111,246 bp in size with a high A+T content of 64.8% and codes for 118 putative open reading frames(ORFs). It contains 37 conserved baculovirus core ORFs, 13 unique ORFs, 26 ORFs that were found in all Lepidoptera baculoviruses and 42 common ORFs. The analysis of nucleotide sequence repeats revealed that the CnmeGV genome differs from the rest of sequenced GVs by a 23 kb and a 17 kb gene block inversions, and does not contain any typical homologous region(hr) except for a region of non-hr-like sequence. Chitinase and cathepsin genes, which are reported to have major roles in the liquefaction of the hosts, were not found in the CnmeGV genome, which explains why CnmeGV infected insects do not show the phenotype of typical liquefaction. Phylogenetic analysis,based on the 37 core baculovirus genes, indicates that CnmeGV is closely related to Adoxophyes orana granulovirus. The genome analysis would contribute to the functional research of CnmeGV,and would benefit to the utilization of CnmeGV as pest control reagent for rice production.

  15. Genome sequence and analysis of Lactobacillus helveticus

    Directory of Open Access Journals (Sweden)

    Paola eCremonesi

    2013-01-01

    Full Text Available The microbiological characterization of lactobacilli is historically well developed, but the genomic analysis is recent. Because of the widespread use of L. helveticus in cheese technology, information concerning the heterogeneity in this species is accumulating rapidly. Recently, the genome of five L. helveticus strains was sequenced to completion and compared with other genomically characterized lactobacilli. The genomic analysis of the first sequenced strain, L. helveticus DPC 4571, isolated from cheese and selected for its characteristics of rapid lysis and high proteolytic activity, has revealed a plethora of genes with industrial potential including those responsible for key metabolic functions such as proteolysis, lipolysis, and cell lysis. These genes and their derived enzymes can facilitate the production of cheese and cheese derivatives with potential for use as ingredients in consumer foods. In addition, L. helveticus has the potential to produce peptides with a biological function, such as angiotensin converting enzyme (ACE inhibitory activity, in fermented dairy products, demonstrating the therapeutic value of this species. A most intriguing feature of the genome of L. helveticus is the remarkable similarity in gene content with many intestinal lactobacilli. Comparative genomics has allowed the identification of key gene sets that facilitate a variety of lifestyles including adaptation to food matrices or the gastrointestinal tract.As genome sequence and functional genomic information continues to explode, key features of the genomes of L. helveticus strains continue to be discovered, answering many questions but also raising many new ones.

  16. The de novo transcriptome and its analysis in the worldwide vegetable pest, Delia antiqua (Diptera: Anthomyiidae).

    Science.gov (United States)

    Zhang, Yu-Juan; Hao, Youjin; Si, Fengling; Ren, Shuang; Hu, Ganyu; Shen, Li; Chen, Bin

    2014-03-10

    The onion maggot Delia antiqua is a major insect pest of cultivated vegetables, especially the onion, and a good model to investigate the molecular mechanisms of diapause. To better understand the biology and diapause mechanism of the insect pest species, D. antiqua, the transcriptome was sequenced using Illumina paired-end sequencing technology. Approximately 54 million reads were obtained, trimmed, and assembled into 29,659 unigenes, with an average length of 607 bp and an N50 of 818 bp. Among these unigenes, 21,605 (72.8%) were annotated in the public databases. All unigenes were then compared against Drosophila melanogaster and Anopheles gambiae. Codon usage bias was analyzed and 332 simple sequence repeats (SSRs) were detected in this organism. These data represent the most comprehensive transcriptomic resource currently available for D. antiqua and will facilitate the study of genetics, genomics, diapause, and further pest control of D. antiqua.

  17. De novo characterization of Larix gmelinii (Rupr.) Rupr. transcriptome and analysis of its gene expression induced by jasmonates.

    Science.gov (United States)

    Men, Lina; Yan, Shanchun; Liu, Guanjun

    2013-08-13

    Larix gmelinii is a dominant tree species in China's boreal forests and plays an important role in the coniferous ecosystem. It is also one of the most economically important tree species in the Chinese timber industry due to excellent water resistance and anti-corrosion of its wood products. Unfortunately, in Northeast China, L. gmelinii often suffers from serious attacks by diseases and insects. The application of exogenous volatile semiochemicals may induce and enhance its resistance against insect or disease attacks; however, little is known regarding the genes and molecular mechanisms related to induced resistance. We performed de novo sequencing and assembly of the L. gmelinii transcriptome using a short read sequencing technology (Illumina). Chemical defenses of L. gmelinii seedlings were induced with jasmonic acid (JA) or methyl jasmonate (MeJA) for 6 hours. Transcriptomes were compared between seedlings induced by JA, MeJA and untreated controls using a tag-based digital gene expression profiling system. In a single run, 25,977,782 short reads were produced and 51,157 unigenes were obtained with a mean length of 517 nt. We sequenced 3 digital gene expression libraries and generated between 3.5 and 5.9 million raw tags, and obtained 52,040 reliable reference genes after removing redundancy. The expression of disease/insect-resistance genes (e.g., phenylalanine ammonialyase, coumarate 3-hydroxylase, lipoxygenase, allene oxide synthase and allene oxide cyclase) was up-regulated. The expression profiles of some abundant genes under different elicitor treatment were studied by using real-time qRT-PCR.The results showed that the expression levels of disease/insect-resistance genes in the seedling samples induced by JA and MeJA were higher than those in the control group. The seedlings induced with MeJA elicited the strongest increases in disease/insect-resistance genes. Both JA and MeJA induced seedlings of L. gmelinii showed significantly increased expression

  18. Pig genome sequence - analysis and publication strategy

    NARCIS (Netherlands)

    Archibald, A.L.; Bolund, L.; Churcher, C.; Fredholm, M.; Groenen, M.A.M.; Harlizius, B.

    2010-01-01

    Background - The pig genome is being sequenced and characterised under the auspices of the Swine Genome Sequencing Consortium. The sequencing strategy followed a hybrid approach combining hierarchical shotgun sequencing of BAC clones and whole genome shotgun sequencing. Results - Assemblies of the B

  19. Multilocus sequence analysis (MLSA) in prokaryotic taxonomy.

    Science.gov (United States)

    Glaeser, Stefanie P; Kämpfer, Peter

    2015-06-01

    To obtain a higher resolution of the phylogenetic relationships of species within a genus or genera within a family, multilocus sequence analysis (MLSA) is currently a widely used method. In MLSA studies, partial sequences of genes coding for proteins with conserved functions ('housekeeping genes') are used to generate phylogenetic trees and subsequently deduce phylogenies. However, MLSA is not only suggested as a phylogenetic tool to support and clarify the resolution of bacterial species with a higher resolution, as in 16S rRNA gene-based studies, but has also been discussed as a replacement for DNA-DNA hybridization (DDH) in species delineation. Nevertheless, despite the fact that MLSA has become an accepted and widely used method in prokaryotic taxonomy, no common generally accepted recommendations have been devised to date for either the whole area of microbial taxonomy or for taxa-specific applications of individual MLSA schemes. The different ways MLSA is performed can vary greatly for the selection of genes, their number, and the calculation method used when comparing the sequences obtained. Here, we provide an overview of the historical development of MLSA and critically review its current application in prokaryotic taxonomy by highlighting the advantages and disadvantages of the method's numerous variations. This provides a perspective for its future use in forthcoming genome-based genotypic taxonomic analyses. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. RNA-Seq analysis and gene discovery of Andrias davidianus using Illumina short read sequencing.

    Directory of Open Access Journals (Sweden)

    Fenggang Li

    Full Text Available The Chinese giant salamander, Andrias davidianus, is an important species in the course of evolution; however, there is insufficient genomic data in public databases for understanding its immunologic mechanisms. High-throughput transcriptome sequencing is necessary to generate an enormous number of transcript sequences from A. davidianus for gene discovery. In this study, we generated more than 40 million reads from samples of spleen and skin tissue using the Illumina paired-end sequencing technology. De novo assembly yielded 87,297 transcripts with a mean length of 734 base pairs (bp. Based on the sequence similarities, searching with known proteins, 38,916 genes were identified. Gene enrichment analysis determined that 981 transcripts were assigned to the immune system. Tissue-specific expression analysis indicated that 443 of transcripts were specifically expressed in the spleen and skin. Among these transcripts, 147 transcripts were found to be involved in immune responses and inflammatory reactions, such as fucolectin, β-defensins and lymphotoxin beta. Eight tissue-specific genes were selected for validation using real time reverse transcription quantitative PCR (qRT-PCR. The results showed that these genes were significantly more expressed in spleen and skin than in other tissues, suggesting that these genes have vital roles in the immune response. This work provides a comprehensive genomic sequence resource for A. davidianus and lays the foundation for future research on the immunologic and disease resistance mechanisms of A. davidianus and other amphibians.

  1. Comparative analysis of sequences from PT 2013

    DEFF Research Database (Denmark)

    Mikkelsen, Susie Sommer

    . All but one sequence mapped to the MCP gene while the last sequence mapped to the Neurofilament gene. Approx. half of the sequences contained no errors while the rest differed with 88-99 percent similarity with most having 99% similarity. One sequence, when BLASTed, showed most similarity to European...... Sheatfish and not EHNV. Generally, mistakes occurred at the ends of the sequences. This can be due to several factors. One is that the sequence has not been trimmed of the sequence primer sites. Another is the lack of quality control of the chromatogram. Finally, sequencing in just one direction can result...

  2. Whole-genome sequencing and genetic variant analysis of a Quarter Horse mare.

    Science.gov (United States)

    Doan, Ryan; Cohen, Noah D; Sawyer, Jason; Ghaffari, Noushin; Johnson, Charlie D; Dindot, Scott V

    2012-02-17

    The catalog of genetic variants in the horse genome originates from a few select animals, the majority originating from the Thoroughbred mare used for the equine genome sequencing project. The purpose of this study was to identify genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs) in the genome of an individual Quarter Horse mare sequenced by next-generation sequencing. Using massively parallel paired-end sequencing, we generated 59.6 Gb of DNA sequence from a Quarter Horse mare resulting in an average of 24.7X sequence coverage. Reads were mapped to approximately 97% of the reference Thoroughbred genome. Unmapped reads were de novo assembled resulting in 19.1 Mb of new genomic sequence in the horse. Using a stringent filtering method, we identified 3.1 million SNPs, 193 thousand INDELs, and 282 CNVs. Genetic variants were annotated to determine their impact on gene structure and function. Additionally, we genotyped this Quarter Horse for mutations of known diseases and for variants associated with particular traits. Functional clustering analysis of genetic variants revealed that most of the genetic variation in the horse's genome was enriched in sensory perception, signal transduction, and immunity and defense pathways. This is the first sequencing of a horse genome by next-generation sequencing and the first genomic sequence of an individual Quarter Horse mare. We have increased the catalog of genetic variants for use in equine genomics by the addition of novel SNPs, INDELs, and CNVs. The genetic variants described here will be a useful resource for future studies of genetic variation regulating performance traits and diseases in equids.

  3. Whole-Genome sequencing and genetic variant analysis of a quarter Horse mare

    Directory of Open Access Journals (Sweden)

    Doan Ryan

    2012-02-01

    Full Text Available Abstract Background The catalog of genetic variants in the horse genome originates from a few select animals, the majority originating from the Thoroughbred mare used for the equine genome sequencing project. The purpose of this study was to identify genetic variants, including single nucleotide polymorphisms (SNPs, insertion/deletion polymorphisms (INDELs, and copy number variants (CNVs in the genome of an individual Quarter Horse mare sequenced by next-generation sequencing. Results Using massively parallel paired-end sequencing, we generated 59.6 Gb of DNA sequence from a Quarter Horse mare resulting in an average of 24.7X sequence coverage. Reads were mapped to approximately 97% of the reference Thoroughbred genome. Unmapped reads were de novo assembled resulting in 19.1 Mb of new genomic sequence in the horse. Using a stringent filtering method, we identified 3.1 million SNPs, 193 thousand INDELs, and 282 CNVs. Genetic variants were annotated to determine their impact on gene structure and function. Additionally, we genotyped this Quarter Horse for mutations of known diseases and for variants associated with particular traits. Functional clustering analysis of genetic variants revealed that most of the genetic variation in the horse's genome was enriched in sensory perception, signal transduction, and immunity and defense pathways. Conclusions This is the first sequencing of a horse genome by next-generation sequencing and the first genomic sequence of an individual Quarter Horse mare. We have increased the catalog of genetic variants for use in equine genomics by the addition of novel SNPs, INDELs, and CNVs. The genetic variants described here will be a useful resource for future studies of genetic variation regulating performance traits and diseases in equids.

  4. Whole-genome sequencing and genetic variant analysis of a Quarter Horse mare.

    KAUST Repository

    Doan, Ryan

    2012-02-17

    BACKGROUND: The catalog of genetic variants in the horse genome originates from a few select animals, the majority originating from the Thoroughbred mare used for the equine genome sequencing project. The purpose of this study was to identify genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs) in the genome of an individual Quarter Horse mare sequenced by next-generation sequencing. RESULTS: Using massively parallel paired-end sequencing, we generated 59.6 Gb of DNA sequence from a Quarter Horse mare resulting in an average of 24.7X sequence coverage. Reads were mapped to approximately 97% of the reference Thoroughbred genome. Unmapped reads were de novo assembled resulting in 19.1 Mb of new genomic sequence in the horse. Using a stringent filtering method, we identified 3.1 million SNPs, 193 thousand INDELs, and 282 CNVs. Genetic variants were annotated to determine their impact on gene structure and function. Additionally, we genotyped this Quarter Horse for mutations of known diseases and for variants associated with particular traits. Functional clustering analysis of genetic variants revealed that most of the genetic variation in the horse\\'s genome was enriched in sensory perception, signal transduction, and immunity and defense pathways. CONCLUSIONS: This is the first sequencing of a horse genome by next-generation sequencing and the first genomic sequence of an individual Quarter Horse mare. We have increased the catalog of genetic variants for use in equine genomics by the addition of novel SNPs, INDELs, and CNVs. The genetic variants described here will be a useful resource for future studies of genetic variation regulating performance traits and diseases in equids.

  5. FAST: FAST Analysis of Sequences Toolbox.

    Science.gov (United States)

    Lawrence, Travis J; Kauffman, Kyle T; Amrine, Katherine C H; Carper, Dana L; Lee, Raymond S; Becich, Peter J; Canales, Claudia J; Ardell, David H

    2015-01-01

    FAST (FAST Analysis of Sequences Toolbox) provides simple, powerful open source command-line tools to filter, transform, annotate and analyze biological sequence data. Modeled after the GNU (GNU's Not Unix) Textutils such as grep, cut, and tr, FAST tools such as fasgrep, fascut, and fastr make it easy to rapidly prototype expressive bioinformatic workflows in a compact and generic command vocabulary. Compact combinatorial encoding of data workflows with FAST commands can simplify the documentation and reproducibility of bioinformatic protocols, supporting better transparency in biological data science. Interface self-consistency and conformity with conventions of GNU, Matlab, Perl, BioPerl, R, and GenBank help make FAST easy and rewarding to learn. FAST automates numerical, taxonomic, and text-based sorting, selection and transformation of sequence records and alignment sites based on content, index ranges, descriptive tags, annotated features, and in-line calculated analytics, including composition and codon usage. Automated content- and feature-based extraction of sites and support for molecular population genetic statistics make FAST useful for molecular evolutionary analysis. FAST is portable, easy to install and secure thanks to the relative maturity of its Perl and BioPerl foundations, with stable releases posted to CPAN. Development as well as a publicly accessible Cookbook and Wiki are available on the FAST GitHub repository at https://github.com/tlawrence3/FAST. The default data exchange format in FAST is Multi-FastA (specifically, a restriction of BioPerl FastA format). Sanger and Illumina 1.8+ FastQ formatted files are also supported. FAST makes it easier for non-programmer biologists to interactively investigate and control biological data at the speed of thought.

  6. FAST: FAST Analysis of Sequences Toolbox

    Directory of Open Access Journals (Sweden)

    Travis J. Lawrence

    2015-05-01

    Full Text Available FAST (FAST Analysis of Sequences Toolbox provides simple, powerful open source command-line tools to filter, transform, annotate and analyze biological sequence data. Modeled after the GNU (GNU’s Not Unix Textutils such as grep, cut, and tr, FAST tools such as fasgrep, fascut, and fastr make it easy to rapidly prototype expressive bioinformatic workflows in a compact and generic command vocabulary. Compact combinatorial encoding of data workflows with FAST commands can simplify the documentation and reproducibility of bioinformatic protocols, supporting better transparency in biological data science. Interface self-consistency and conformity with conventions of GNU, Matlab, Perl, BioPerl, R and GenBank help make FAST easy and rewarding to learn. FAST automates numerical, taxonomic, and text-based sorting, selection and transformation of sequence records and alignment sites based on content, index ranges, descriptive tags, annotated features, and in-line calculated analytics, including composition and codon usage. Automated content- and feature-based extraction of sites and support for molecular population genetic statistics makes FAST useful for molecular evolutionary analysis. FAST is portable, easy to install and secure thanks to the relative maturity of its Perl and BioPerl foundations, with stable releases posted to CPAN. Development as well as a publicly accessible Cookbook and Wiki are available on the FAST GitHub repository at https://github.com/tlawrence3/FAST. The default data exchange format in FAST is Multi-FastA (specifically, a restriction of BioPerl FastA format. Sanger and Illumina 1.8+ FastQ formatted files are also supported. FAST makes it easier for non-programmer biologists to interactively investigate and control biological data at the speed of thought.

  7. De Novo Assembly of the Donkey White Blood Cell Transcriptome and a Comparative Analysis of Phenotype-Associated Genes between Donkeys and Horses.

    Science.gov (United States)

    Xie, Feng-Yun; Feng, Yu-Long; Wang, Hong-Hui; Ma, Yun-Feng; Yang, Yang; Wang, Yin-Chao; Shen, Wei; Pan, Qing-Jie; Yin, Shen; Sun, Yu-Jiang; Ma, Jun-Yu

    2015-01-01

    Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus) for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR) protein database. We also compared the donkey protein sequences with those of the horse (E. caballus) and wild horse (E. przewalskii), and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement.

  8. De Novo Assembly of the Donkey White Blood Cell Transcriptome and a Comparative Analysis of Phenotype-Associated Genes between Donkeys and Horses.

    Directory of Open Access Journals (Sweden)

    Feng-Yun Xie

    Full Text Available Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR protein database. We also compared the donkey protein sequences with those of the horse (E. caballus and wild horse (E. przewalskii, and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement.

  9. A basic analysis toolkit for biological sequences

    Directory of Open Access Journals (Sweden)

    Siragusa Enrico

    2007-09-01

    Full Text Available Abstract This paper presents a software library, nicknamed BATS, for some basic sequence analysis tasks. Namely, local alignments, via approximate string matching, and global alignments, via longest common subsequence and alignments with affine and concave gap cost functions. Moreover, it also supports filtering operations to select strings from a set and establish their statistical significance, via z-score computation. None of the algorithms is new, but although they are generally regarded as fundamental for sequence analysis, they have not been implemented in a single and consistent software package, as we do here. Therefore, our main contribution is to fill this gap between algorithmic theory and practice by providing an extensible and easy to use software library that includes algorithms for the mentioned string matching and alignment problems. The library consists of C/C++ library functions as well as Perl library functions. It can be interfaced with Bioperl and can also be used as a stand-alone system with a GUI. The software is available at http://www.math.unipa.it/~raffaele/BATS/ under the GNU GPL.

  10. Whole genome sequence analysis of Mycobacterium suricattae

    KAUST Repository

    Dippenaar, Anzaan

    2015-10-21

    Tuberculosis occurs in various mammalian hosts and is caused by a range of different lineages of the Mycobacterium tuberculosis complex (MTBC). A recently described member, Mycobacterium suricattae, causes tuberculosis in meerkats (Suricata suricatta) in Southern Africa and preliminary genetic analysis showed this organism to be closely related to an MTBC pathogen of rock hyraxes (Procavia capensis), the dassie bacillus. Here we make use of whole genome sequencing to describe the evolution of the genome of M. suricattae, including known and novel regions of difference, SNPs and IS6110 insertion sites. We used genome-wide phylogenetic analysis to show that M. suricattae clusters with the chimpanzee bacillus, previously isolated from a chimpanzee (Pan troglodytes) in West Africa. We propose an evolutionary scenario for the Mycobacterium africanum lineage 6 complex, showing the evolutionary relationship of M. africanum and chimpanzee bacillus, and the closely related members M. suricattae, dassie bacillus and Mycobacterium mungi.

  11. Facile analysis and sequencing of linear and branched peptide boronic acids by MALDI mass spectrometry.

    Science.gov (United States)

    B Crumpton, Jason; Zhang, Wenyu; L Santos, Webster

    2011-05-01

    Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time-consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable, and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries.

  12. Transcriptome analysis of the mud crab (Scylla paramamosain by 454 deep sequencing: assembly, annotation, and marker discovery.

    Directory of Open Access Journals (Sweden)

    Hongyu Ma

    Full Text Available In this study, we reported the characterization of the first transcriptome of the mud crab (Scylla paramamosain. Pooled cDNAs of four tissue types from twelve wild individuals were sequenced using the Roche 454 FLX platform. Analysis performed included de novo assembly of transcriptome sequences, functional annotation, and molecular marker discovery. A total of 1,314,101 high quality reads with an average length of 411 bp were generated by 454 sequencing on a mixed cDNA library. De novo assembly of these 1,314,101 reads produced 76,778 contigs (consisting of 818,154 reads with 5.4-fold average sequencing coverage. The remaining 495,947 reads were singletons. A total of 78,268 unigenes were identified based on sequence similarity with known proteins (E≤0.00001 in UniProt and non-redundant protein databases. Meanwhile, 44,433 sequences were identified (E≤0.00001 using a BLASTN search against the NCBI nucleotide database. Gene Ontology (GO analysis indicated that biosynthetic process, cell part, and ion binding were the most abundant terms in biological process, cellular component, and molecular function categories, respectively. Kyoto Encyclopedia of Genes and Genome (KEGG pathway analysis revealed that 4,878 unigenes distributed in 281 different pathways. In addition, 19,011 microsatellites and 37,063 potential single nucleotide polymorphisms were detected from the transcriptome of S. paramamosain. Finally, thirty polymorphic microsatellite markers were developed and used to assess genetic diversity of a wild population of S. paramamosain. So far, existing sequence resources for S. paramamosain are extremely limited. The present study provides a characterization of transcriptome from multiple tissues and individuals, as well as an assessment of genetic diversity of a wild population. These sequence resources will facilitate the investigation of population genetic diversity, the development of genetic maps, and the conduct of molecular marker

  13. Pig genome sequence - analysis and publication strategy

    DEFF Research Database (Denmark)

    Archibald, Alan L.; Bolund, Lars; Churcher, Carol;

    2010-01-01

    BACKGROUND: The pig genome is being sequenced and characterised under the auspices of the Swine Genome Sequencing Consortium. The sequencing strategy followed a hybrid approach combining hierarchical shotgun sequencing of BAC clones and whole genome shotgun sequencing. RESULTS: Assemblies......) is under construction and will incorporate whole genome shotgun sequence (WGS) data providing > 30x genome coverage. The WGS sequence, most of which comprise short Illumina/Solexa reads, were generated from DNA from the same single Duroc sow as the source of the BAC library from which clones were...

  14. The simple fool's guide to population genomics via RNA-Seq: An introduction to high-throughput sequencing data analysis

    DEFF Research Database (Denmark)

    De Wit, P.; Pespeni, M.H.; Ladner, J.T.;

    2012-01-01

    to Population Genomics via RNA-seq' (SFG), a document intended to serve as an easy-to-follow protocol, walking a user through one example of high-throughput sequencing data analysis of nonmodel organisms. It is by no means an exhaustive protocol, but rather serves as an introduction to the bioinformatic methods......://sfg.stanford.edu, that includes detailed protocols for data processing and analysis, along with a repository of custom-made scripts and sample files. Steps included in the SFG range from tissue collection to de novo assembly, blast annotation, alignment, gene expression, functional enrichment, SNP detection, principal components...

  15. De novo assembly and comparative analysis of root transcriptomes from different varieties of Panax ginseng C. A. Meyer grown in different environments.

    Science.gov (United States)

    Zhen, Gang; Zhang, Lei; Du, YaNan; Yu, RenBo; Liu, XinMin; Cao, FangRui; Chang, Qi; Deng, XingWang; Xia, Mian; He, Hang

    2015-11-01

    Panax ginseng C. A. Meyer is an important traditional herb in eastern Asia. It contains ginsenosides, which are primary bioactive compounds with medicinal properties. Although ginseng has been cultivated since at least the Ming dynasty to increase production, cultivated ginseng has lower quantities of ginsenosides and lower disease resistance than ginseng grown under natural conditions. We extracted root RNA from six varieties of fifth-year P. ginseng cultivars representing four different growth conditions, and performed Illumina paired-end sequencing. In total, 163,165,706 raw reads were obtained and used to generate a de novo transcriptome that consisted of 151,763 contigs (76,336 unigenes), of which 100,648 contigs (66.3%) were successfully annotated. Differential expression analysis revealed that most differentially expressed genes (DEGs) were upregulated (246 out of 258, 95.3%) in ginseng grown under natural conditions compared with that grown under artificial conditions. These DEGs were enriched in gene ontology (GO) terms including response to stimuli and localization. In particular, some key ginsenoside biosynthesis-related genes, including HMG-CoA synthase (HMGS), mevalonate kinase (MVK), and squalene epoxidase (SE), were upregulated in wild-grown ginseng. Moreover, a high proportion of disease resistance-related genes were upregulated in wild-grown ginseng. This study is the first transcriptome analysis to compare wild-grown and cultivated ginseng, and identifies genes that may produce higher ginsenoside content and better disease resistance in the wild; these genes may have the potential to improve cultivated ginseng grown in artificial environments.

  16. MIDDAS-M: motif-independent de novo detection of secondary metabolite gene clusters through the integration of genome sequencing and transcriptome data.

    Science.gov (United States)

    Umemura, Myco; Koike, Hideaki; Nagano, Nozomi; Ishii, Tomoko; Kawano, Jin; Yamane, Noriko; Kozone, Ikuko; Horimoto, Katsuhisa; Shin-ya, Kazuo; Asai, Kiyoshi; Yu, Jiujiang; Bennett, Joan W; Machida, Masayuki

    2013-01-01

    Many bioactive natural products are produced as "secondary metabolites" by plants, bacteria, and fungi. During the middle of the 20th century, several secondary metabolites from fungi revolutionized the pharmaceutical industry, for example, penicillin, lovastatin, and cyclosporine. They are generally biosynthesized by enzymes encoded by clusters of coordinately regulated genes, and several motif-based methods have been developed to detect secondary metabolite biosynthetic (SMB) gene clusters using the sequence information of typical SMB core genes such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). However, no detection method exists for SMB gene clusters that are functional and do not include core SMB genes at present. To advance the exploration of SMB gene clusters, especially those without known core genes, we developed MIDDAS-M, a motif-independent de novodetection algorithm for SMB gene clusters. We integrated virtual gene cluster generation in an annotated genome sequence with highly sensitive scoring of the cooperative transcriptional regulation of cluster member genes. MIDDAS-M accurately predicted 38 SMB gene clusters that have been experimentally confirmed and/or predicted by other motif-based methods in 3 fungal strains. MIDDAS-M further identified a new SMB gene cluster for ustiloxin B, which was experimentally validated. Sequence analysis of the cluster genes indicated a novel mechanism for peptide biosynthesis independent of NRPS. Because it is fully computational and independent of empirical knowledge about SMB core genes, MIDDAS-M allows a large-scale, comprehensive analysis of SMB gene clusters, including those with novel biosynthetic mechanisms that do not contain any functionally characterized genes.

  17. Bilateral renal agenesis/hypoplasia/dysplasia (BRAHD: postmortem analysis of 45 cases with breakpoint mapping of two de novo translocations.

    Directory of Open Access Journals (Sweden)

    Louise Harewood

    Full Text Available BACKGROUND: Bilateral renal agenesis/hypoplasia/dysplasia (BRAHD is a relatively common, lethal malformation in humans. Established clinical risk factors include maternal insulin dependent diabetes mellitus and male sex of the fetus. In the majority of cases, no specific etiology can be established, although teratogenic, syndromal and single gene causes can be assigned to some cases. METHODOLOGY/PRINCIPAL FINDINGS: 45 unrelated fetuses, stillbirths or infants with lethal BRAHD were ascertained through a single regional paediatric pathology service (male:female 34:11 or 3.1:1. The previously reported phenotypic overlaps with VACTERL, caudal dysgenesis, hemifacial microsomia and Müllerian defects were confirmed. A new finding is that 16/45 (35.6%; m:f 13:3 or 4.3:1 BRAHD cases had one or more extrarenal malformations indicative of a disoder of laterality determination including; incomplete lobulation of right lung (seven cases, malrotation of the gut (seven cases and persistence of the left superior vena cava (five cases. One such case with multiple laterality defects and sirelomelia was found to have a de novo apparently balanced reciprocal translocation 46,XY,t(2;6(p22.3;q12. Translocation breakpoint mapping was performed by interphase fluorescent in-situ hybridization (FISH using nuclei extracted from archival tissue sections in both this case and an isolated bilateral renal agenesis case associated with a de novo 46,XY,t(1;2(q41;p25.3. Both t(2;6 breakpoints mapped to gene-free regions with no strong evidence of cis-regulatory potential. Ten genes localized within 500 kb of the t(1;2 breakpoints. Wholemount in-situ expression analyses of the mouse orthologs of these genes in embryonic mouse kidneys showed strong expression of Esrrg, encoding a nuclear steroid hormone receptor. Immunohistochemical analysis showed that Esrrg was restricted to proximal ductal tissue within the embryonic kidney. CONCLUSIONS/SIGNIFICANCE: The previously unreported

  18. Multilocus sequence analysis of the family Halomonadaceae.

    Science.gov (United States)

    de la Haba, Rafael R; Márquez, M Carmen; Papke, R Thane; Ventosa, Antonio

    2012-03-01

    Multilocus sequence analysis (MLSA) protocols have been developed for species circumscription for many taxa. However, at present, no studies based on MLSA have been performed within any moderately halophilic bacterial group. To test the usefulness of MLSA with these kinds of micro-organisms, the family Halomonadaceae, which includes mainly halophilic bacteria, was chosen as a model. This family comprises ten genera with validly published names and 85 species of environmental, biotechnological and clinical interest. In some cases, the phylogenetic relationships between members of this family, based on 16S rRNA gene sequence comparisons, are not clear and a deep phylogenetic analysis using several housekeeping genes seemed appropriate. Here, MLSA was applied using the 16S rRNA, 23S rRNA, atpA, gyrB, rpoD and secA genes for species of the family Halomonadaceae. Phylogenetic trees based on the individual and concatenated gene sequences revealed that the family Halomonadaceae formed a monophyletic group of micro-organisms within the order Oceanospirillales. With the exception of the genera Halomonas and Modicisalibacter, all other genera within this family were phylogenetically coherent. Five of the six studied genes (16S rRNA, 23S rRNA, gyrB, rpoD and secA) showed a consistent evolutionary history. However, the results obtained with the atpA gene were different; thus, this gene may not be considered useful as an individual gene phylogenetic marker within this family. The phylogenetic methods produced variable results, with those generated from the maximum-likelihood and neighbour-joining algorithms being more similar than those obtained by maximum-parsimony methods. Horizontal gene transfer (HGT) plays an important evolutionary role in the family Halomonadaceae; however, the impact of recombination events in the phylogenetic analysis was minimized by concatenating the six loci, which agreed with the current taxonomic scheme for this family. Finally, the findings of

  19. Combinatorial analysis and algorithms for quasispecies reconstruction using next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Vincenti Donatella

    2011-01-01

    Full Text Available Abstract Background Next-generation sequencing (NGS offers a unique opportunity for high-throughput genomics and has potential to replace Sanger sequencing in many fields, including de-novo sequencing, re-sequencing, meta-genomics, and characterisation of infectious pathogens, such as viral quasispecies. Although methodologies and software for whole genome assembly and genome variation analysis have been developed and refined for NGS data, reconstructing a viral quasispecies using NGS data remains a challenge. This application would be useful for analysing intra-host evolutionary pathways in relation to immune responses and antiretroviral therapy exposures. Here we introduce a set of formulae for the combinatorial analysis of a quasispecies, given a NGS re-sequencing experiment and an algorithm for quasispecies reconstruction. We require that sequenced fragments are aligned against a reference genome, and that the reference genome is partitioned into a set of sliding windows (amplicons. The reconstruction algorithm is based on combinations of multinomial distributions and is designed to minimise the reconstruction of false variants, called in-silico recombinants. Results The reconstruction algorithm was applied to error-free simulated data and reconstructed a high percentage of true variants, even at a low genetic diversity, where the chance to obtain in-silico recombinants is high. Results on empirical NGS data from patients infected with hepatitis B virus, confirmed its ability to characterise different viral variants from distinct patients. Conclusions The combinatorial analysis provided a description of the difficulty to reconstruct a quasispecies, given a determined amplicon partition and a measure of population diversity. The reconstruction algorithm showed good performance both considering simulated data and real data, even in presence of sequencing errors.

  20. RIKEN Integrated Sequence Analysis (RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer

    Science.gov (United States)

    Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Nagaoka, Sumiharu; Sasaki, Nobuya; Carninci, Piero; Konno, Hideaki; Akiyama, Junichi; Nishi, Katsuo; Kitsunai, Tokuji; Tashiro, Hideo; Itoh, Mari; Sumi, Noriko; Ishii, Yoshiyuki; Nakamura, Shin; Hazama, Makoto; Nishine, Tsutomu; Harada, Akira; Yamamoto, Rintaro; Matsumoto, Hiroyuki; Sakaguchi, Sumito; Ikegami, Takashi; Kashiwagi, Katsuya; Fujiwake, Syuji; Inoue, Kouji; Togawa, Yoshiyuki; Izawa, Masaki; Ohara, Eiji; Watahiki, Masanori; Yoneda, Yuko; Ishikawa, Tomokazu; Ozawa, Kaori; Tanaka, Takumi; Matsuura, Shuji; Kawai, Jun; Okazaki, Yasushi; Muramatsu, Masami; Inoue, Yorinao; Kira, Akira; Hayashizaki, Yoshihide

    2000-01-01

    The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3′ end and 5′ end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can

  1. RIKEN integrated sequence analysis (RISA) system--384-format sequencing pipeline with 384 multicapillary sequencer.

    Science.gov (United States)

    Shibata, K; Itoh, M; Aizawa, K; Nagaoka, S; Sasaki, N; Carninci, P; Konno, H; Akiyama, J; Nishi, K; Kitsunai, T; Tashiro, H; Itoh, M; Sumi, N; Ishii, Y; Nakamura, S; Hazama, M; Nishine, T; Harada, A; Yamamoto, R; Matsumoto, H; Sakaguchi, S; Ikegami, T; Kashiwagi, K; Fujiwake, S; Inoue, K; Togawa, Y

    2000-11-01

    The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3' end and 5' end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be

  2. SVAMP: Sequence variation analysis, maps and phylogeny

    KAUST Repository

    Naeem, Raeece

    2014-04-03

    Summary: SVAMP is a stand-alone desktop application to visualize genomic variants (in variant call format) in the context of geographical metadata. Users of SVAMP are able to generate phylogenetic trees and perform principal coordinate analysis in real time from variant call format (VCF) and associated metadata files. Allele frequency map, geographical map of isolates, Tajima\\'s D metric, single nucleotide polymorphism density, GC and variation density are also available for visualization in real time. We demonstrate the utility of SVAMP in tracking a methicillin-resistant Staphylococcus aureus outbreak from published next-generation sequencing data across 15 countries. We also demonstrate the scalability and accuracy of our software on 245 Plasmodium falciparum malaria isolates from three continents. Availability and implementation: The Qt/C++ software code, binaries, user manual and example datasets are available at http://cbrc.kaust.edu.sa/svamp. © The Author 2014.

  3. Statistical analysis of next generation sequencing data

    CERN Document Server

    Nettleton, Dan

    2014-01-01

    Next Generation Sequencing (NGS) is the latest high throughput technology to revolutionize genomic research. NGS generates massive genomic datasets that play a key role in the big data phenomenon that surrounds us today. To extract signals from high-dimensional NGS data and make valid statistical inferences and predictions, novel data analytic and statistical techniques are needed. This book contains 20 chapters written by prominent statisticians working with NGS data. The topics range from basic preprocessing and analysis with NGS data to more complex genomic applications such as copy number variation and isoform expression detection. Research statisticians who want to learn about this growing and exciting area will find this book useful. In addition, many chapters from this book could be included in graduate-level classes in statistical bioinformatics for training future biostatisticians who will be expected to deal with genomic data in basic biomedical research, genomic clinical trials and personalized med...

  4. Time fluctuation analysis of forest fire sequences

    Science.gov (United States)

    Vega Orozco, Carmen D.; Kanevski, Mikhaïl; Tonini, Marj; Golay, Jean; Pereira, Mário J. G.

    2013-04-01

    Forest fires are complex events involving both space and time fluctuations. Understanding of their dynamics and pattern distribution is of great importance in order to improve the resource allocation and support fire management actions at local and global levels. This study aims at characterizing the temporal fluctuations of forest fire sequences observed in Portugal, which is the country that holds the largest wildfire land dataset in Europe. This research applies several exploratory data analysis measures to 302,000 forest fires occurred from 1980 to 2007. The applied clustering measures are: Morisita clustering index, fractal and multifractal dimensions (box-counting), Ripley's K-function, Allan Factor, and variography. These algorithms enable a global time structural analysis describing the degree of clustering of a point pattern and defining whether the observed events occur randomly, in clusters or in a regular pattern. The considered methods are of general importance and can be used for other spatio-temporal events (i.e. crime, epidemiology, biodiversity, geomarketing, etc.). An important contribution of this research deals with the analysis and estimation of local measures of clustering that helps understanding their temporal structure. Each measure is described and executed for the raw data (forest fires geo-database) and results are compared to reference patterns generated under the null hypothesis of randomness (Poisson processes) embedded in the same time period of the raw data. This comparison enables estimating the degree of the deviation of the real data from a Poisson process. Generalizations to functional measures of these clustering methods, taking into account the phenomena, were also applied and adapted to detect time dependences in a measured variable (i.e. burned area). The time clustering of the raw data is compared several times with the Poisson processes at different thresholds of the measured function. Then, the clustering measure value

  5. Movement Pattern Analysis Based on Sequence Signatures

    Directory of Open Access Journals (Sweden)

    Seyed Hossein Chavoshi

    2015-09-01

    Full Text Available Increased affordability and deployment of advanced tracking technologies have led researchers from various domains to analyze the resulting spatio-temporal movement data sets for the purpose of knowledge discovery. Two different approaches can be considered in the analysis of moving objects: quantitative analysis and qualitative analysis. This research focuses on the latter and uses the qualitative trajectory calculus (QTC, a type of calculus that represents qualitative data on moving point objects (MPOs, and establishes a framework to analyze the relative movement of multiple MPOs. A visualization technique called sequence signature (SESI is used, which enables to map QTC patterns in a 2D indexed rasterized space in order to evaluate the similarity of relative movement patterns of multiple MPOs. The applicability of the proposed methodology is illustrated by means of two practical examples of interacting MPOs: cars on a highway and body parts of a samba dancer. The results show that the proposed method can be effectively used to analyze interactions of multiple MPOs in different domains.

  6. A unified test of linkage analysis and rare-variant association for analysis of pedigree sequence data.

    Science.gov (United States)

    Hu, Hao; Roach, Jared C; Coon, Hilary; Guthery, Stephen L; Voelkerding, Karl V; Margraf, Rebecca L; Durtschi, Jacob D; Tavtigian, Sean V; Shankaracharya; Wu, Wilfred; Scheet, Paul; Wang, Shuoguo; Xing, Jinchuan; Glusman, Gustavo; Hubley, Robert; Li, Hong; Garg, Vidu; Moore, Barry; Hood, Leroy; Galas, David J; Srivastava, Deepak; Reese, Martin G; Jorde, Lynn B; Yandell, Mark; Huff, Chad D

    2014-07-01

    High-throughput sequencing of related individuals has become an important tool for studying human disease. However, owing to technical complexity and lack of available tools, most pedigree-based sequencing studies rely on an ad hoc combination of suboptimal analyses. Here we present pedigree-VAAST (pVAAST), a disease-gene identification tool designed for high-throughput sequence data in pedigrees. pVAAST uses a sequence-based model to perform variant and gene-based linkage analysis. Linkage information is then combined with functional prediction and rare variant case-control association information in a unified statistical framework. pVAAST outperformed linkage and rare-variant association tests in simulations and identified disease-causing genes from whole-genome sequence data in three human pedigrees with dominant, recessive and de novo inheritance patterns. The approach is robust to incomplete penetrance and locus heterogeneity and is applicable to a wide variety of genetic traits. pVAAST maintains high power across studies of monogenic, high-penetrance phenotypes in a single pedigree to highly polygenic, common phenotypes involving hundreds of pedigrees.

  7. De Novo Transcriptome Analysis of the Common New Zealand Stick Insect Clitarchus hookeri (Phasmatodea Reveals Genes Involved in Olfaction, Digestion and Sexual Reproduction.

    Directory of Open Access Journals (Sweden)

    Chen Wu

    Full Text Available Phasmatodea, more commonly known as stick insects, have been poorly studied at the molecular level for several key traits, such as components of the sensory system and regulators of reproduction and development, impeding a deeper understanding of their functional biology. Here, we employ de novo transcriptome analysis to identify genes with primary functions related to female odour reception, digestion, and male sexual traits in the New Zealand common stick insect Clitarchus hookeri (White. The female olfactory gene repertoire revealed ten odorant binding proteins with three recently duplicated, 12 chemosensory proteins, 16 odorant receptors, and 17 ionotropic receptors. The majority of these olfactory genes were over-expressed in female antennae and have the inferred function of odorant reception. Others that were predominantly expressed in male terminalia (n = 3 and female midgut (n = 1 suggest they have a role in sexual reproduction and digestion, respectively. Over-represented transcripts in the midgut were enriched with digestive enzyme gene families. Clitarchus hookeri is likely to harbour nine members of an endogenous cellulase family (glycoside hydrolase family 9, two of which appear to be specific to the C. hookeri lineage. All of these cellulase sequences fall into four main phasmid clades and show gene duplication events occurred early in the diversification of Phasmatodea. In addition, C. hookeri genome is likely to express γ-proteobacteria pectinase transcripts that have recently been shown to be the result of horizontal transfer. We also predicted 711 male terminalia-enriched transcripts that are candidate accessory gland proteins, 28 of which were annotated to have molecular functions of peptidase activity and peptidase inhibitor activity, two groups being widely reported to regulate female reproduction through proteolytic cascades. Our study has yielded new insights into the genetic basis of odour detection, nutrient digestion

  8. De Novo Assembly and Analysis of Tartary Buckwheat (Fagopyrum tataricum Garetn. Transcriptome Discloses Key Regulators Involved in Salt-Stress Response

    Directory of Open Access Journals (Sweden)

    Qi Wu

    2017-10-01

    Full Text Available Soil salinization has been a tremendous obstacle for agriculture production. The regulatory networks underlying salinity adaption in model plants have been extensively explored. However, limited understanding of the salt response mechanisms has hindered the planting and production in Fagopyrum tataricum, an economic and health-beneficial plant mainly distributing in southwest China. In this study, we performed physiological analysis and found that salt stress of 200 mM NaCl solution significantly affected the relative water content (RWC, electrolyte leakage (EL, malondialdehyde (MDA content, peroxidase (POD and superoxide dismutase (SOD activities in tartary buckwheat seedlings. Further, we conducted transcriptome comparison between control and salt treatment to identify potential regulatory components involved in F. tataricum salt responses. A total of 53.15 million clean reads from control and salt-treated libraries were produced via an Illumina sequencing approach. Then we de novo assembled these reads into a transcriptome dataset containing 57,921 unigenes with N50 length of 1400 bp and total length of 44.5 Mb. A total of 36,688 unigenes could find matches in public databases. GO, KEGG and KOG classification suggested the enrichment of these unigenes in 56 sub-categories, 25 KOG, and 273 pathways, respectively. Comparison of the transcriptome expression patterns between control and salt treatment unveiled 455 differentially expressed genes (DEGs. Further, we found the genes encoding for protein kinases, phosphatases, heat shock proteins (HSPs, ATP-binding cassette (ABC transporters, glutathione S-transferases (GSTs, abiotic-related transcription factors and circadian clock might be relevant to the salinity adaption of this species. Thus, this study offers an insight into salt tolerance mechanisms, and will serve as useful genetic information for tolerant elite breeding programs in future.

  9. De novo analysis of receptor binding affinity data of xanthine adenosine receptor antagonists.

    Science.gov (United States)

    Dalpiaz, A; Gardenghi, A; Borea, P A

    1995-03-01

    The receptor binding affinity data to adenosine A1 and A2 receptors of a wide series of xanthine derivatives have been analyzed by means of the Free-Wilson model. The analysis of the individual group contribution shows, for both A1 and A2 receptors, the primary importance of the presence of bulky substituents at position 8 for an optimum receptor binding. Moreover, considering the different aij contributions of bulky substituents at position 8 for affinity to A1 with respect to A2 receptors, this position appears to be the most important for the synthesis of highly A1 selective xanthine derivatives. Moreover the analysis of group contributions for other substitution positions of the xanthine moiety allows to state that suitable substitutions at positions 3 and 7 could confer some degree of A2 selectivity.

  10. A De Novo-Assembly Based Data Analysis Pipeline for Plant Obligate Parasite Metatranscriptomic Studies

    OpenAIRE

    Li Guo; Kelly S Allen; Greg A Deiulio; Yong Zhang; Madeiras, Angela M.; Robert L Wick; Li-Jun Ma

    2016-01-01

    Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression anal...

  11. Schlieren sequence analysis using computer vision

    Science.gov (United States)

    Smith, Nathanial Timothy

    Computer vision-based methods are proposed for extraction and measurement of flow structures of interest in schlieren video. As schlieren data has increased with faster frame rates, we are faced with thousands of images to analyze. This presents an opportunity to study global flow structures over time that may not be evident from surface measurements. A degree of automation is desirable to extract flow structures and features to give information on their behavior through the sequence. Using an interdisciplinary approach, the analysis of large schlieren data is recast as a computer vision problem. The double-cone schlieren sequence is used as a testbed for the methodology; it is unique in that it contains 5,000 images, complex phenomena, and is feature rich. Oblique structures such as shock waves and shear layers are common in schlieren images. A vision-based methodology is used to provide an estimate of oblique structure angles through the unsteady sequence. The methodology has been applied to a complex flowfield with multiple shocks. A converged detection success rate between 94% and 97% for these structures is obtained. The modified curvature scale space is used to define features at salient points on shock contours. A challenge in developing methods for feature extraction in schlieren images is the reconciliation of existing techniques with features of interest to an aerodynamicist. Domain-specific knowledge of physics must therefore be incorporated into the definition and detection phases. Known location and physically possible structure representations form a knowledge base that provides a unique feature definition and extraction. Model tip location and the motion of a shock intersection across several thousand frames are identified, localized, and tracked. Images are parsed into physically meaningful labels using segmentation. Using this representation, it is shown that in the double-cone flowfield, the dominant unsteady motion is associated with large scale

  12. Integrative analysis of circadian transcriptome and metabolic network reveals the role of de novo purine synthesis in circadian control of cell cycle.

    Science.gov (United States)

    Li, Ying; Li, Guang; Görling, Benjamin; Luy, Burkhard; Du, Jiulin; Yan, Jun

    2015-02-01

    Metabolism is the major output of the circadian clock in many organisms. We developed a computational method to integrate both circadian gene expression and metabolic network. Applying this method to zebrafish circadian transcriptome, we have identified large clusters of metabolic genes containing mostly genes in purine and pyrimidine metabolism in the metabolic network showing similar circadian phases. Our metabolomics analysis found that the level of inosine 5'-monophosphate (IMP), an intermediate metabolite in de novo purine synthesis, showed significant circadian oscillation in larval zebrafish. We focused on IMP dehydrogenase (impdh), a rate-limiting enzyme in de novo purine synthesis, with three circadian oscillating gene homologs: impdh1a, impdh1b and impdh2. Functional analysis revealed that impdh2 contributes to the daily rhythm of S phase in the cell cycle while impdh1a contributes to ocular development and pigment synthesis. The three zebrafish homologs of impdh are likely regulated by different circadian transcription factors. We propose that the circadian regulation of de novo purine synthesis that supplies crucial building blocks for DNA replication is an important mechanism conferring circadian rhythmicity on the cell cycle. Our method is widely applicable to study the impact of circadian transcriptome on metabolism in complex organisms.

  13. BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in upland cotton.

    Directory of Open Access Journals (Sweden)

    Ramesh Buyyarapu

    Full Text Available Although new and emerging next-generation sequencing (NGS technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.. Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg. To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.

  14. Development of Transcriptomic Markers for Population Analysis Using Restriction Site Associated RNA Sequencing (RARseq.

    Directory of Open Access Journals (Sweden)

    Magdy S Alabady

    Full Text Available We describe restriction site associated RNA sequencing (RARseq, an RNAseq-based genotype by sequencing (GBS method. It includes the construction of RNAseq libraries from double stranded cDNA digested with selected restriction enzymes. To test this, we constructed six single- and six-dual-digested RARseq libraries from six F2 pitcher plant individuals and sequenced them on a half of a Miseq run. On average, the de novo approach of population genome analysis detected 544 and 570 RNA SNPs, whereas the reference transcriptome-based approach revealed an average of 1907 and 1876 RNA SNPs per individual, from single- and dual-digested RARseq data, respectively. The average numbers of RNA SNPs and alleles per loci are 1.89 and 2.17, respectively. Our results suggest that the RARseq protocol allows good depth of coverage per loci for detecting RNA SNPs and polymorphic loci for population genomics and mapping analyses. In non-model systems where complete genomes sequences are not always available, RARseq data can be analyzed in reference to the transcriptome. In addition to enriching for functional markers, this method may prove particularly useful in organisms where the genomes are not favorable for DNA GBS.

  15. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing

    Science.gov (United States)

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S.; Perkins, David L.

    2016-01-01

    The human microbiome has emerged as a major player in regulating human health and disease. Translation studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using shotgun whole genome sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1×106 reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that shotgun whole genome sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection. PMID:26718401

  16. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing.

    Science.gov (United States)

    Ranjan, Ravi; Rani, Asha; Metwally, Ahmed; McGee, Halvor S; Perkins, David L

    2016-01-22

    The human microbiome has emerged as a major player in regulating human health and disease. Translational studies of the microbiome have the potential to indicate clinical applications such as fecal transplants and probiotics. However, one major issue is accurate identification of microbes constituting the microbiota. Studies of the microbiome have frequently utilized sequencing of the conserved 16S ribosomal RNA (rRNA) gene. We present a comparative study of an alternative approach using whole genome shotgun sequencing (WGS). In the present study, we analyzed the human fecal microbiome compiling a total of 194.1 × 10(6) reads from a single sample using multiple sequencing methods and platforms. Specifically, after establishing the reproducibility of our methods with extensive multiplexing, we compared: 1) The 16S rRNA amplicon versus the WGS method, 2) the Illumina HiSeq versus MiSeq platforms, 3) the analysis of reads versus de novo assembled contigs, and 4) the effect of shorter versus longer reads. Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.

  17. Analysis of mutations in the entire coding sequence of the factor VIII gene

    Energy Technology Data Exchange (ETDEWEB)

    Bidichadani, S.I.; Lanyon, W.G.; Connor, J.M. [Glascow Univ. (United Kingdom)] [and others

    1994-09-01

    Hemophilia A is a common X-linked recessive disorder of bleeding caused by deleterious mutations in the gene for clotting factor VIII. The large size of the factor VIII gene, the high frequency of de novo mutations and its tissue-specific expression complicate the detection of mutations. We have used a combination of RT-PCR of ectopic factor VIII transcripts and genomic DNA-PCRs to amplify the entire essential sequence of the factor VIII gene. This is followed by chemical mismatch cleavage analysis and direct sequencing in order to facilitate a comprehensive search for mutations. We describe the characterization of nine potentially pathogenic mutations, six of which are novel. In each case, a correlation of the genotype with the observed phenotype is presented. In order to evaluate the pathogenicity of the five missense mutations detected, we have analyzed them for evolutionary sequence conservation and for their involvement of sequence motifs catalogued in the PROSITE database of protein sites and patterns.

  18. An 11bp region with stem formation potential is essential for de novo DNA methylation of the RPS element.

    Directory of Open Access Journals (Sweden)

    Matthew Gentry

    Full Text Available The initiation of DNA methylation in Arabidopsis is controlled by the RNA-directed DNA methylation (RdDM pathway that uses 24nt siRNAs to recruit de novo methyltransferase DRM2 to the target site. We previously described the REPETITIVE PETUNIA SEQUENCE (RPS fragment that acts as a hot spot for de novo methylation, for which it requires the cooperative activity of all three methyltransferases MET1, CMT3 and DRM2, but not the RdDM pathway. RPS contains two identical 11nt elements in inverted orientation, interrupted by a 18nt spacer, which resembles the features of a stemloop structure. The analysis of deletion/substitution derivatives of this region showed that deletion of one 11nt element RPS is sufficient to eliminate de novo methylation of RPS. In addition, deletion of a 10nt region directly adjacent to one of the 11nt elements, significantly reduced de novo methylation. When both 11nt regions were replaced by two 11nt elements with altered DNA sequence but unchanged inverted repeat homology, DNA methylation was not affected, indicating that de novo methylation was not targeted to a specific DNA sequence element. These data suggest that de novo DNA methylation is attracted by a secondary structure to which the two 11nt elements contribute, and that the adjacent 10nt region influences the stability of this structure. This resembles the recognition of structural features by DNA methyltransferases in animals and suggests that similar mechanisms exist in plants.

  19. Whole genome duplication and enrichment of metal cation transporters revealed by de novo genome sequencing of extremely halotolerant black yeast Hortaea werneckii.

    Directory of Open Access Journals (Sweden)

    Metka Lenassi

    Full Text Available Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern. Two of its most striking characteristics that may be associated with a halotolerant lifestyle are the large genetic redundancy and the expansion of genes encoding metal cation transporters. Although no sexual state of H. werneckii has yet been described, a mating locus with characteristics of heterothallic fungi was found. The total assembly size of the genome is 51.6 Mb, larger than most phylogenetically related fungi, coding for almost twice the usual number of predicted genes (23333. The genome appears to have experienced a relatively recent whole genome duplication, and contains two highly identical gene copies of almost every protein. This is consistent with some previous studies that reported increases in genomic DNA content triggered by exposure to salt stress. In hypersaline conditions transmembrane ion transport is of utmost importance. The analysis of predicted metal cation transporters showed that most types of transporters experienced several gene duplications at various points during their evolution. Consequently they are present in much higher numbers than expected. The resulting diversity of transporters presents interesting biotechnological opportunities for improvement of halotolerance of salt-sensitive species. The involvement of plasma P-type H⁺ ATPases in adaptation to different concentrations of salt was indicated by their salt dependent transcription. This was not the case with vacuolar H⁺ ATPases, which were transcribed constitutively. The availability of this genomic sequence is expected to promote the research of H. werneckii. Studying its extreme halotolerance will not only contribute to our understanding of life in hypersaline

  20. Transcriptome sequencing and differential gene expression analysis in Viola yedoensis Makino (Fam. Violaceae) responsive to cadmium (Cd) pollution

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jian [Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute of Sichuan Agricultural University, Wenjiang, Sichuan (China); Luo, Mao [Drug Discovery Research Center of Luzhou Medical College, Luzhou, Sichuan (China); Zhu, Ye; He, Ying; Wang, Qin [Department of Pharmacy of Luzhou Medical College, Luzhou, Sichuan (China); Zhang, Chun, E-mail: zc83good@126.com [Department of Pharmacy of Luzhou Medical College, Luzhou, Sichuan (China)

    2015-03-27

    Viola yedoensis Makino is an important Chinese traditional medicine plant adapted to cadmium (Cd) pollution regions. Illumina sequencing technology was used to sequence the transcriptome of V. yedoensis Makino. We sequenced Cd-treated (VIYCd) and untreated (VIYCK) samples of V. yedoensis, and obtained 100,410,834 and 83,587,676 high quality reads, respectively. After de novo assembly and quantitative assessment, 109,800 unigenes were finally generated with an average length of 661 bp. We then obtained functional annotations by aligning unigenes with public protein databases including NR, NT, SwissProt, KEGG and COG. In addition, 892 differentially expressed genes (DEGs) were investigated between the two libraries of untreated (VIYCK) and Cd-treated (VIYCd) plants. Moreover, 15 randomly selected DEGs were further validated with qRT-PCR and the results were highly accordant with the Solexa analysis. This study firstly generated a successful global analysis of the V. yedoensis transcriptome and it will provide for further studies on gene expression, genomics, and functional genomics in Violaceae. - Highlights: • A de novo assembly generated 109,800 unigenes and 5,4479 of them were annotated. • 31,285 could be classified into 26 COG categories. • 263 biosynthesis pathways were predicted and classified into five categories. • 892 DEGs were detected and 15 of them were validated by qRT-PCR.

  1. De novo assembly of highly diverse viral populations

    Directory of Open Access Journals (Sweden)

    Yang Xiao

    2012-09-01

    Full Text Available Abstract Background Extensive genetic diversity in viral populations within infected hosts and the divergence of variants from existing reference genomes impede the analysis of deep viral sequencing data. A de novo population consensus assembly is valuable both as a single linear representation of the population and as a backbone on which intra-host variants can be accurately mapped. The availability of consensus assemblies and robustly mapped variants are crucial to the genetic study of viral disease progression, transmission dynamics, and viral evolution. Existing de novo assembly techniques fail to robustly assemble ultra-deep sequence data from genetically heterogeneous populations such as viruses into full-length genomes due to the presence of extensive genetic variability, contaminants, and variable sequence coverage. Results We present VICUNA, a de novo assembly algorithm suitable for generating consensus assemblies from genetically heterogeneous populations. We demonstrate its effectiveness on Dengue, Human Immunodeficiency and West Nile viral populations, representing a range of intra-host diversity. Compared to state-of-the-art assemblers designed for haploid or diploid systems, VICUNA recovers full-length consensus and captures insertion/deletion polymorphisms in diverse samples. Final assemblies maintain a high base calling accuracy. VICUNA program is publicly available at: http://www.broadinstitute.org/scientific-community/science/projects/viral-genomics/ viral-genomics-analysis-software. Conclusions We developed VICUNA, a publicly available software tool, that enables consensus assembly of ultra-deep sequence derived from diverse viral populations. While VICUNA was developed for the analysis of viral populations, its application to other heterogeneous sequence data sets such as metagenomic or tumor cell population samples may prove beneficial in these fields of research.

  2. Electrochemical measurement for analysis of DNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Cho, S.B.; Hong, J.S.; Pak, J.H. [Korea University, Seoul (Korea); Kim, Y.M. [National Institute of Health, Seoul (Korea)

    2002-02-01

    One of the important roles of a DNA chip is the capability of detecting genetic diseases and mutations by analyzing DNA sequence. For a successful electrochemical genotyping, several aspects should be considered including the chemical treatment of electrode surface, DNA immobilization on electrode, hybridization, choice of an intercalator to be selectively bound to double standed DNA, and an equipment for detecting and analyzing the output singal. Au was used as the electrode material, 2-mercaptoethanol was used for linking DNA to Au electrode, and methylene blue was used as an indicator that can be bound to a double stranded DNA selectively. From the analysis of reductive current of this indicator that was bound to a double stranded DNA on an electrode, a normal double stranded DNA was able to be distinguished from a single stranded DNA in just a few seconds. Also, it was found that the peak reduction current of indicator is proportional to the concentration of target DNA to be hybridized with probe DNA. Therefore, it is possible to realize a simple and cheap DNA sensor using the electrochemical measurement for genotyping. (author). 20 refs., 8 figs., 1 tab.

  3. Genome sequence and comparative analysis of Avibacterium paragallinarum

    Science.gov (United States)

    Requena, David; Chumbe, Ana; Torres, Michael; Alzamora, Ofelia; Ramirez, Manuel; Valdivia-Olarte, Hugo; Gutierrez, Andres Hazaet; Izquierdo-Lara, Ray; Saravia, Luis Enrique; Zavaleta, Milagros; Tataje-Lavanda, Luis; Best, Ivan; Fernández-Sánchez, Manolo; Icochea, Eliana; Zimic, Mirko; Fernández-Díaz, Manolo

    2013-01-01

    Background: Avibacterium paragallinarum, the causative agent of infectious coryza, is a highly contagious respiratory acute disease of poultry, which affects commercial chickens, laying hens and broilers worldwide. Methodology: In this study, we performed the whole genome sequencing, assembly and annotation of a Peruvian isolate of A. paragallinarum. Genome was sequenced in a 454 GS FLX Titanium system. De novo assembly was performed and annotation was completed with GS De Novo Assembler 2.6 using the H. influenzae str. F3031 gene model. Manual curation of the genome was performed with Artemis. Putative function of genes was predicted with Blast2GO. Virulence factors were identified by comparison with the Virulence Factor Database. Results: The genome obtained has a length of 2.47 Mb with 40.66% of GC content. Seventy five large contigs (>500 nt) were obtained, which comprised 1,204 predicted genes. All the contigs are available in Genbank [GenBank: PRJNA64665]. A total of 103 virulence factors, reported in the Virulence Factor Database, were found in A. paragallinarum. Forty four of them are present in 7 species of Haemophilus, which are related with pathogenesis, virulence and host immune system evasion. A tetracycline-resistance associated transposon (Tn10), was found in A. paragallinarum, possibly acting as a defense mechanism. Discussion and conclusion: The availability of A. paragallinarum genome represents an important source of information for the development of diagnostic tests, genotyping, and novel antigens for potential vaccines against infectious coryza. Identification of virulence factors contributes to better understanding the pathogenesis, and planning efforts for prevention and control of the disease. PMID:23861570

  4. Novel algorithms for protein sequence analysis

    NARCIS (Netherlands)

    Ye, Kai

    2008-01-01

    Each protein is characterized by its unique sequential order of amino acids, the so-called protein sequence. Biology”s paradigm is that this order of amino acids determines the protein”s architecture and function. In this thesis, we introduce novel algorithms to analyze protein sequences. Chapter 1

  5. Pig genome sequence - analysis and publication strategy

    DEFF Research Database (Denmark)

    Archibald, Alan L.; Bolund, Lars; Churcher, Carol

    2010-01-01

    preferentially selected for sequencing. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement the data have been released into public sequence repositories (Genbank/EMBL, NCBI/Ensembl trace repositories) in a timely manner and in advance of publication. CONCLUSIONS...

  6. A time- and cost-effective strategy to sequence mammalian Y Chromosomes: an application to the de novo assembly of gorilla Y

    OpenAIRE

    Tomaszkiewicz, Marta; Rangavittal, Samarth; Cechova, Monika; Sanchez, Rebeca Campos; Fescemyer, Howard W.; Harris, Robert; Ye, Danling; O'Brien, Patricia C.M.; Chikhi, Rayan; Ryder, Oliver A; Malcolm A Ferguson-Smith; Medvedev, Paul; Makova, Kateryna D.

    2016-01-01

    The mammalian Y Chromosome sequence, critical for studying male fertility and dispersal, is enriched in repeats and palindromes, and thus, is the most difficult component of the genome to assemble. Previously, expensive and labor-intensive BAC-based techniques were used to sequence the Y for a handful of mammalian species. Here, we present a much faster and more affordable strategy for sequencing and assembling mammalian Y Chromosomes of sufficient quality for most comparative genomics analys...

  7. The DNA sequence, annotation and analysis of human chromosome 3

    DEFF Research Database (Denmark)

    Muzny, Donna M; Scherer, Steven E; Kaul, Rajinder

    2006-01-01

    After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chr...

  8. Sequencing and Analysis of a Genomic Fragment Provide an Insight into the Dunaliella viridis Genomic Sequence

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ming SUN; Yuan-Ping TANG; Xiang-Zong MENG; Wen-Wen ZHANG; Shan LI; Zhi-Rui DENG; Zheng-Kai XU; Ren-Tao SONG

    2006-01-01

    Dunaliella is a genus of wall-less unicellular eukaryotic green alga. Its exceptional resistances to salt and various other stresses have made it an ideal model for stress tolerance study. However, very little is known about its genome and genomic sequences. In this study, we sequenced and analyzed a 29,268 bp genomic fragment from Dunaliella viridis. The fragment showed low sequence homology to the GenBank database. At the nucleotide level, only a segment with significant sequence homology to 18S rRNA was found. The fragment contained six putative genes, but only one gene showed significant homology at the protein level to GenBank database. The average GC content of this sequence was 51.1%, which was much lower than that of close related green algae Chlamydomonas (65.7%). Significant segmental duplications were found within this fragment. The duplicated sequences accounted for about 35.7% of the entire region. Large amounts of simple sequence repeats (microsatellites) were found, with strong bias towards (AC)n type (76%). Analysis of other Dunaliella genomic sequences in the GenBank database (total 25,749 bp) was in agreement with these findings. These sequence features made it difficult to sequence Dunaliella genomic sequences. Further investigation should be made to reveal the biological significance of these unique sequence features.

  9. From de novo mutations to personalized therapeutic interventions in autism.

    Science.gov (United States)

    Brandler, William M; Sebat, Jonathan

    2015-01-01

    The high heritability, early age at onset, and reproductive disadvantages of autism spectrum disorders (ASDs) are consistent with an etiology composed of dominant-acting de novo (spontaneous) mutations. Mutation detection by microarray analysis and DNA sequencing has confirmed that de novo copy-number variants or point mutations in protein-coding regions of genes contribute to risk, and some of the underlying causal variants and genes have been identified. As our understanding of autism genes develops, the spectrum of autism is breaking up into quanta of many different genetic disorders. Given the diversity of etiologies and underlying biochemical pathways, personalized therapy for ASDs is logical, and clinical genetic testing is a prerequisite.

  10. A time- and cost-effective strategy to sequence mammalian Y Chromosomes: an application to the de novo assembly of gorilla Y.

    Science.gov (United States)

    Tomaszkiewicz, Marta; Rangavittal, Samarth; Cechova, Monika; Campos Sanchez, Rebeca; Fescemyer, Howard W; Harris, Robert; Ye, Danling; O'Brien, Patricia C M; Chikhi, Rayan; Ryder, Oliver A; Ferguson-Smith, Malcolm A; Medvedev, Paul; Makova, Kateryna D

    2016-04-01

    The mammalian Y Chromosome sequence, critical for studying male fertility and dispersal, is enriched in repeats and palindromes, and thus, is the most difficult component of the genome to assemble. Previously, expensive and labor-intensive BAC-based techniques were used to sequence the Y for a handful of mammalian species. Here, we present a much faster and more affordable strategy for sequencing and assembling mammalian Y Chromosomes of sufficient quality for most comparative genomics analyses and for conservation genetics applications. The strategy combines flow sorting, short- and long-read genome and transcriptome sequencing, and droplet digital PCR with novel and existing computational methods. It can be used to reconstruct sex chromosomes in a heterogametic sex of any species. We applied our strategy to produce a draft of the gorilla Y sequence. The resulting assembly allowed us to refine gene content, evaluate copy number of ampliconic gene families, locate species-specific palindromes, examine the repetitive element content, and produce sequence alignments with human and chimpanzee Y Chromosomes. Our results inform the evolution of the hominine (human, chimpanzee, and gorilla) Y Chromosomes. Surprisingly, we found the gorilla Y Chromosome to be similar to the human Y Chromosome, but not to the chimpanzee Y Chromosome. Moreover, we have utilized the assembled gorilla Y Chromosome sequence to design genetic markers for studying the male-specific dispersal of this endangered species.

  11. GATA: a graphic alignment tool for comparative sequence analysis

    Directory of Open Access Journals (Sweden)

    Nix David A

    2005-01-01

    Full Text Available Abstract Background Several problems exist with current methods used to align DNA sequences for comparative sequence analysis. Most dynamic programming algorithms assume that conserved sequence elements are collinear. This assumption appears valid when comparing orthologous protein coding sequences. Functional constraints on proteins provide strong selective pressure against sequence inversions, and minimize sequence duplications and feature shuffling. For non-coding sequences this collinearity assumption is often invalid. For example, enhancers contain clusters of transcription factor binding sites that change in number, orientation, and spacing during evolution yet the enhancer retains its activity. Dot plot analysis is often used to estimate non-coding sequence relatedness. Yet dot plots do not actually align sequences and thus cannot account well for base insertions or deletions. Moreover, they lack an adequate statistical framework for comparing sequence relatedness and are limited to pairwise comparisons. Lastly, dot plots and dynamic programming text outputs fail to provide an intuitive means for visualizing DNA alignments. Results To address some of these issues, we created a stand alone, platform independent, graphic alignment tool for comparative sequence analysis (GATA http://gata.sourceforge.net/. GATA uses the NCBI-BLASTN program and extensive post-processing to identify all small sub-alignments above a low cut-off score. These are graphed as two shaded boxes, one for each sequence, connected by a line using the coordinate system of their parent sequence. Shading and colour are used to indicate score and orientation. A variety of options exist for querying, modifying and retrieving conserved sequence elements. Extensive gene annotation can be added to both sequences using a standardized General Feature Format (GFF file. Conclusions GATA uses the NCBI-BLASTN program in conjunction with post-processing to exhaustively align two DNA

  12. Full sequence analysis of the original Sapporo virus.

    Science.gov (United States)

    Nakanishi, Kaori; Tatsumi, Masatoshi; Kinoshita-Numata, Kazuko; Tsugawa, Takeshi; Nakata, Shuji; Tsutsumi, Hiroyuki

    2011-09-01

    In this study, the full-length genome sequence of the prototype of sapovirus, namely Sapporo virus (SV82), was identified. Sapporo virus RNA was extracted from a fecal sample, amplified by RT-PCR and the PCR products sequenced directly and analyzed. Sequence analysis showed that Sapporo virus consists of 7433 nucleotides and has three open reading frames. The Sapporo strain shows 91.7% nucleotide sequence identity to the Manchester virus. Phylogenic analysis has also revealed the closeness of Sapporo virus to other sapovirus/genogroup I strains. Basic information on the evolutionary history of sapovirus analysis is provided here.

  13. SeqHBase: a big data toolset for family based sequencing data analysis.

    Science.gov (United States)

    He, Min; Person, Thomas N; Hebbring, Scott J; Heinzen, Ethan; Ye, Zhan; Schrodi, Steven J; McPherson, Elizabeth W; Lin, Simon M; Peissig, Peggy L; Brilliant, Murray H; O'Rawe, Jason; Robison, Reid J; Lyon, Gholson J; Wang, Kai

    2015-04-01

    Whole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies are increasingly used to identify disease-contributing mutations in human genomic studies. It can be a significant challenge to process such data, especially when a large family or cohort is sequenced. Our objective was to develop a big data toolset to efficiently manipulate genome-wide variants, functional annotations and coverage, together with conducting family based sequencing data analysis. Hadoop is a framework for reliable, scalable, distributed processing of large data sets using MapReduce programming models. Based on Hadoop and HBase, we developed SeqHBase, a big data-based toolset for analysing family based sequencing data to detect de novo, inherited homozygous, or compound heterozygous mutations that may contribute to disease manifestations. SeqHBase takes as input BAM files (for coverage at every site), variant call format (VCF) files (for variant calls) and functional annotations (for variant prioritisation). We applied SeqHBase to a 5-member nuclear family and a 10-member 3-generation family with WGS data, as well as a 4-member nuclear family with WES data. Analysis times were almost linearly scalable with number of data nodes. With 20 data nodes, SeqHBase took about 5 secs to analyse WES familial data and approximately 1 min to analyse WGS familial data. These results demonstrate SeqHBase's high efficiency and scalability, which is necessary as WGS and WES are rapidly becoming standard methods to study the genetics of familial disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  14. Indel variant analysis of short-read sequencing data with Scalpel.

    Science.gov (United States)

    Fang, Han; Bergmann, Ewa A; Arora, Kanika; Vacic, Vladimir; Zody, Michael C; Iossifov, Ivan; O'Rawe, Jason A; Wu, Yiyang; Jimenez Barron, Laura T; Rosenbaum, Julie; Ronemus, Michael; Lee, Yoon-Ha; Wang, Zihua; Dikoglu, Esra; Jobanputra, Vaidehi; Lyon, Gholson J; Wigler, Michael; Schatz, Michael C; Narzisi, Giuseppe

    2016-12-01

    As the second most common type of variation in the human genome, insertions and deletions (indels) have been linked to many diseases, but the discovery of indels of more than a few bases in size from short-read sequencing data remains challenging. Scalpel (http://scalpel.sourceforge.net) is an open-source software for reliable indel detection based on the microassembly technique. It has been successfully used to discover mutations in novel candidate genes for autism, and it is extensively used in other large-scale studies of human diseases. This protocol gives an overview of the algorithm and describes how to use Scalpel to perform highly accurate indel calling from whole-genome and whole-exome sequencing data. We provide detailed instructions for an exemplary family-based de novo study, but we also characterize the other two supported modes of operation: single-sample and somatic analysis. Indel normalization, visualization and annotation of the mutations are also illustrated. Using a standard server, indel discovery and characterization in the exonic regions of the example sequencing data can be completed in ∼5 h after read mapping.

  15. Targeted Next-Generation Sequencing Analysis of 1,000 Individuals with Intellectual Disability.

    Science.gov (United States)

    Grozeva, Detelina; Carss, Keren; Spasic-Boskovic, Olivera; Tejada, Maria-Isabel; Gecz, Jozef; Shaw, Marie; Corbett, Mark; Haan, Eric; Thompson, Elizabeth; Friend, Kathryn; Hussain, Zaamin; Hackett, Anna; Field, Michael; Renieri, Alessandra; Stevenson, Roger; Schwartz, Charles; Floyd, James A B; Bentham, Jamie; Cosgrove, Catherine; Keavney, Bernard; Bhattacharya, Shoumo; Hurles, Matthew; Raymond, F Lucy

    2015-12-01

    To identify genetic causes of intellectual disability (ID), we screened a cohort of 986 individuals with moderate to severe ID for variants in 565 known or candidate ID-associated genes using targeted next-generation sequencing. Likely pathogenic rare variants were found in ∼11% of the cases (113 variants in 107/986 individuals: ∼8% of the individuals had a likely pathogenic loss-of-function [LoF] variant, whereas ∼3% had a known pathogenic missense variant). Variants in SETD5, ATRX, CUL4B, MECP2, and ARID1B were the most common causes of ID. This study assessed the value of sequencing a cohort of probands to provide a molecular diagnosis of ID, without the availability of DNA from both parents for de novo sequence analysis. This modeling is clinically relevant as 28% of all UK families with dependent children are single parent households. In conclusion, to diagnose patients with ID in the absence of parental DNA, we recommend investigation of all LoF variants in known genes that cause ID and assessment of a limited list of proven pathogenic missense variants in these genes. This will provide 11% additional diagnostic yield beyond the 10%-15% yield from array CGH alone.

  16. Discovery, genotyping and characterization of structural variation and novel sequence at single nucleotide resolution from de novo genome assemblies on a population scale

    DEFF Research Database (Denmark)

    Huang, Shujia; Rao, Junhua; Ye, Weijian

    2015-01-01

    Comprehensive recognition of genomic variation in one individual is important for understanding disease and developing personalized medication and treatment. Many tools based on DNA re-sequencing exist for identification of single nucleotide polymorphisms, small insertions and deletions (indels...

  17. Discovery, genotyping and characterization of structural variation and novel sequence at single nucleotide resolution from de novo genome assemblies on a population scale

    DEFF Research Database (Denmark)

    Huang, Shujia; Rao, Junhua; Ye, Weijian

    2015-01-01

    Comprehensive recognition of genomic variation in one individual is important for understanding disease and developing personalized medication and treatment. Many tools based on DNA re-sequencing exist for identification of single nucleotide polymorphisms, small insertions and deletions (indels) ...

  18. Discovery, genotyping and characterization of structural variation and novel sequence at single nucleotide resolution from de novo genome assemblies on a population scale

    DEFF Research Database (Denmark)

    Huang, Shujia; Rao, Junhua; Ye, Weijian;

    2015-01-01

    Comprehensive recognition of genomic variation in one individual is important for understanding disease and developing personalized medication and treatment. Many tools based on DNA re-sequencing exist for identification of single nucleotide polymorphisms, small insertions and deletions (indels) ...

  19. Scalable Kernel Methods and Algorithms for General Sequence Analysis

    Science.gov (United States)

    Kuksa, Pavel

    2011-01-01

    Analysis of large-scale sequential data has become an important task in machine learning and pattern recognition, inspired in part by numerous scientific and technological applications such as the document and text classification or the analysis of biological sequences. However, current computational methods for sequence comparison still lack…

  20. [Tabular excel editor for analysis of aligned nucleotide sequences].

    Science.gov (United States)

    Demkin, V V

    2010-01-01

    Excel platform was used for transition of results of multiple aligned nucleotide sequences obtained using the BLAST network service to the form appropriate for visual analysis and editing. Two macros operators for MS Excel 2007 were constructed. The array of aligned sequences transformed into Excel table and processed using macros operators is more appropriate for analysis than initial html data.

  1. Establishing a framework for comparative analysis of genome sequences

    Energy Technology Data Exchange (ETDEWEB)

    Bansal, A.K.

    1995-06-01

    This paper describes a framework and a high-level language toolkit for comparative analysis of genome sequence alignment The framework integrates the information derived from multiple sequence alignment and phylogenetic tree (hypothetical tree of evolution) to derive new properties about sequences. Multiple sequence alignments are treated as an abstract data type. Abstract operations have been described to manipulate a multiple sequence alignment and to derive mutation related information from a phylogenetic tree by superimposing parsimonious analysis. The framework has been applied on protein alignments to derive constrained columns (in a multiple sequence alignment) that exhibit evolutionary pressure to preserve a common property in a column despite mutation. A Prolog toolkit based on the framework has been implemented and demonstrated on alignments containing 3000 sequences and 3904 columns.

  2. Mathematical methods of analysis of biopolymer sequences

    CERN Document Server

    Gindikin, S G

    1992-01-01

    This collection contains papers by participants in the seminar on mathematical methods in molecular biology who worked for several years at the Laboratory of Molecular Biology and Bioorganic Chemistry (now the Institute of Physical and Chemical Problems in Biology) at Moscow State University. The seminar united mathematicians and biologists around the problems of biological sequences. The collection includes original results as well as expository material and spans a range of perspectives, from purely mathematical problems to algorithms and their computer realizations. For this reason, the book is of interest to mathematicians, statisticians, biologists, and computational scientists who work with biopolymer sequences.

  3. Transcriptome Analysis of the Emerald Ash Borer (EAB), Agrilus planipennis: De Novo Assembly, Functional Annotation and Comparative Analysis.

    Science.gov (United States)

    Duan, Jun; Ladd, Tim; Doucet, Daniel; Cusson, Michel; vanFrankenhuyzen, Kees; Mittapalli, Omprakash; Krell, Peter J; Quan, Guoxing

    2015-01-01

    The Emerald ash borer (EAB), Agrilus planipennis, is an invasive phloem-feeding insect pest of ash trees. Since its initial discovery near the Detroit, US- Windsor, Canada area in 2002, the spread of EAB has had strong negative economic, social and environmental impacts in both countries. Several transcriptomes from specific tissues including midgut, fat body and antenna have recently been generated. However, the relatively low sequence depth, gene coverage and completeness limited the usefulness of these EAB databases. High-throughput deep RNA-Sequencing (RNA-Seq) was used to obtain 473.9 million pairs of 100 bp length paired-end reads from various life stages and tissues. These reads were assembled into 88,907 contigs using the Trinity strategy and integrated into 38,160 unigenes after redundant sequences were removed. We annotated 11,229 unigenes by searching against the public nr, Swiss-Prot and COG. The EAB transcriptome assembly was compared with 13 other sequenced insect species, resulting in the prediction of 536 unigenes that are Coleoptera-specific. Differential gene expression revealed that 290 unigenes are expressed during larval molting and 3,911 unigenes during metamorphosis from larvae to pupae, respectively (FDR2). In addition, 1,167 differentially expressed unigenes were identified from larval and adult midguts, 435 unigenes were up-regulated in larval midgut and 732 unigenes were up-regulated in adult midgut. Most of the genes involved in RNA interference (RNAi) pathways were identified, which implies the existence of a system RNAi in EAB. This study provides one of the most fundamental and comprehensive transcriptome resources available for EAB to date. Identification of the tissue- stage- or species- specific unigenes will benefit the further study of gene functions during growth and metamorphosis processes in EAB and other pest insects.

  4. De novo SNP calling from RAD sequences for a mink (Neovison vison) specific genotyping assay

    DEFF Research Database (Denmark)

    Thirstrup, Janne Pia; Pujolar, José Martin; Larsen, Peter F

    and require a large market to cover the cost. New technologies based on next generation sequencing (NGS) have made it possible to identify thousands of SNPs using a cost effective and fast method. The method can be used for non-model organisms in conservation biology and for production species with small...... population sizes. The aim of this study was to create a mink specific SNP panel well suited for population genetic studies, parental testing and forensic investigations. A SNP panel specific for American mink (Neovison vison) has been generated from Restriction site-Associated DNA (RAD) sequencing. Fourteen...... mink from Brown and Black color types were obtained. A mean of 49,789,860.2 (± 9,813,587.2) raw reads of high quality per sample were sequenced. SNPs were called using the software pipeline Stacks. The populations program was used to estimate population structure and genetic divergence between the two...

  5. Application of wavelet transform in runoff sequence analysis

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A wavelet transform is applied to runoff analysis to obtain the composition of the runoff sequence and to forecast future runoff. An observed runoff sequence is firstly decomposed and reconstructed by wavelet transform and its expanding tendency is derived. Then, the runoff sequence is forecasted by the back propagation artificial neural networks (BPANN) and by a wavelet transform combined with BPANN. The earlier researches seldom involve the problem of how to choose wavelet function, which is important and cannot be ignored when the wavelet transform is used. With application of the developed approach to the analysis of runoff sequence, several kinds of wavelet functions have been tested.

  6. De novo SNP calling from RAD sequences for a mink (Neovison vison) specific genotyping assay

    DEFF Research Database (Denmark)

    Thirstrup, Janne Pia; Pujolar, José Martin; Larsen, Peter F;

    population sizes. The aim of this study was to create a mink specific SNP panel well suited for population genetic studies, parental testing and forensic investigations. A SNP panel specific for American mink (Neovison vison) has been generated from Restriction site-Associated DNA (RAD) sequencing. Fourteen...... and require a large market to cover the cost. New technologies based on next generation sequencing (NGS) have made it possible to identify thousands of SNPs using a cost effective and fast method. The method can be used for non-model organisms in conservation biology and for production species with small...

  7. De novo transcriptomic analysis and development of EST-SSR markers in the Siberian tiger (Panthera tigris altaica).

    Science.gov (United States)

    Lu, Taofeng; Sun, Yujiao; Ma, Qin; Zhu, Minghao; Liu, Dan; Ma, Jianzhang; Ma, Yuehui; Chen, Hongyan; Guan, Weijun

    2016-12-01

    The Siberian tiger, Panthera tigris altaica, is an endangered species, and much more work is needed to protect this species, which is still vulnerable to extinction. Conservation efforts may be supported by the genetic assessment of wild populations, for which highly specific microsatellite markers are required. However, only a limited amount of genetic sequence data is available for this species. To identify the genes involved in the lung transcriptome and to develop additional simple sequence repeat (SSR) markers for the Siberian tiger, we used high-throughput RNA-Seq to characterize the Siberian tiger transcriptome in lung tissue (designated 'PTA-lung') and a pooled tissue sample (designated 'PTA'). Approximately 47.5 % (33,187/69,836) of the lung transcriptome was annotated in four public databases (Nr, Swiss-Prot, KEGG, and COG). The annotated genes formed a potential pool for gene identification in the tiger. An analysis of the genes differentially expressed in the PTA lung, and PTA samples revealed that the tiger may have suffered a series of diseases before death. In total, 1062 non-redundant SSRs were identified in the Siberian tiger transcriptome. Forty-three primer pairs were randomly selected for amplification reactions, and 26 of the 43 pairs were also used to evaluate the levels of genetic polymorphism. Fourteen primer pairs (32.56 %) amplified products that were polymorphic in size in P. tigris altaica. In conclusion, the transcriptome sequences will provide a valuable genomic resource for genetic research, and these new SSR markers comprise a reasonable number of loci for the genetic analysis of wild and captive populations of P. tigris altaica.

  8. Elastic sequence correlation for human action analysis.

    Science.gov (United States)

    Wang, Li; Cheng, Li; Wang, Liang

    2011-06-01

    This paper addresses the problem of automatically analyzing and understanding human actions from video footage. An "action correlation" framework, elastic sequence correlation (ESC), is proposed to identify action subsequences from a database of (possibly long) video sequences that are similar to a given query video action clip. In particular, we show that two well-known algorithms, namely approximate pattern matching in computer and information sciences and dynamic time warping (DTW) method in signal processing, are special cases of our ESC framework. The proposed framework is applied to two important real-world applications: action pattern retrieval, as well as action segmentation and recognition, where, on average, its run time speed (in matlab) is about 3.3 frames per second. In addition, comparing with the state-of-the-art algorithms on a number of challenging data sets, our approach is demonstrated to perform competitively.

  9. Cardiac nonrigid motion analysis from image sequences

    Institute of Scientific and Technical Information of China (English)

    LIU Huafeng

    2006-01-01

    Noninvasive estimation of the soft tissue kinematics properties from medical image sequences has many important clinical and physiological implications, such as the diagnosis of heart diseases and the understanding of cardiac mechanics. In this paper, we present a biomechanics based strategy, framed as a priori constraints for the ill-posed motion recovery problema, to realize estimation of the cardiac motion and deformation parameters. By constructing the heart dynamics system equations from biomechanics principles, we use the finite element method to generate smooth estimates.of heart kinematics throughout the cardiac cycle. We present the application of the strategy to the estimation of displacements and strains from in vivo left ventricular magnetic resonance image sequence.

  10. De novo assembly and characterization of the leaf, bud, and fruit transcriptome from the vulnerable tree Juglans mandshurica for the development of 20 new microsatellite markers using Illumina sequencing.

    Science.gov (United States)

    Hu, Zhuang; Zhang, Tian; Gao, Xiao-Xiao; Wang, Yang; Zhang, Qiang; Zhou, Hui-Juan; Zhao, Gui-Fang; Wang, Ma-Li; Woeste, Keith E; Zhao, Peng

    2016-04-01

    Manchurian walnut (Juglans mandshurica Maxim.) is a vulnerable, temperate deciduous tree valued for its wood and nut, but transcriptomic and genomic data for the species are very limited. Next generation sequencing (NGS) has made it possible to develop molecular markers for this species rapidly and efficiently. Our goal is to use transcriptome information from RNA-Seq to understand development in J. mandshurica and develop polymorphic simple sequence repeats (SSRs, microsatellites) to understand the species' population genetics. In this study, more than 47.7 million clean reads were generated using Illumina sequencing technology. De novo assembly yielded 99,869 unigenes with an average length of 747 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.32 %) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) identified 15,903 (16.9 %) unigenes. Further, we identified and characterized 63 new transcriptome-derived microsatellite markers. By testing the markers on 4 to 14 individuals from four populations, we found that 20 were polymorphic and easily amplified. The number of alleles per locus ranged from 2 to 8. The observed and expected heterozygosity per locus ranged from 0.209 to 0.813 and 0.335 to 0.842, respectively. These twenty microsatellite markers will be useful for studies of population genetics, diversity, and genetic structure, and they will undoubtedly benefit future breeding studies of this walnut species. Moreover, the information uncovered in this research will also serve as a useful genetic resource for understanding the transcriptome and development of J. mandshurica and other Juglans species.

  11. De-novo assembly and analysis of the heterozygous triploid genome of the wine spoilage yeast Dekkera bruxellensis AWRI1499.

    Science.gov (United States)

    Curtin, Chris D; Borneman, Anthony R; Chambers, Paul J; Pretorius, Isak S

    2012-01-01

    Despite its industrial importance, the yeast species Dekkera (Brettanomyces) bruxellensis has remained poorly understood at the genetic level. In this study we describe whole genome sequencing and analysis for a prevalent wine spoilage strain, AWRI1499. The 12.7 Mb assembly, consisting of 324 contigs in 99 scaffolds (super-contigs) at 26-fold coverage, exhibits a relatively high density of single nucleotide polymorphisms (SNPs). Haplotype sampling for 1.2% of open reading frames suggested that the D. bruxellensis AWRI1499 genome is comprised of a moderately heterozygous diploid genome, in combination with a divergent haploid genome. Gene content analysis revealed enrichment in membrane proteins, particularly transporters, along with oxidoreductase enzymes. Availability of this assembly and annotation provides a resource for further investigation of genomic organization in this species, and functional characterization of genes that may confer important phenotypic traits.

  12. De-novo assembly and analysis of the heterozygous triploid genome of the wine spoilage yeast Dekkera bruxellensis AWRI1499.

    Directory of Open Access Journals (Sweden)

    Chris D Curtin

    Full Text Available Despite its industrial importance, the yeast species Dekkera (Brettanomyces bruxellensis has remained poorly understood at the genetic level. In this study we describe whole genome sequencing and analysis for a prevalent wine spoilage strain, AWRI1499. The 12.7 Mb assembly, consisting of 324 contigs in 99 scaffolds (super-contigs at 26-fold coverage, exhibits a relatively high density of single nucleotide polymorphisms (SNPs. Haplotype sampling for 1.2% of open reading frames suggested that the D. bruxellensis AWRI1499 genome is comprised of a moderately heterozygous diploid genome, in combination with a divergent haploid genome. Gene content analysis revealed enrichment in membrane proteins, particularly transporters, along with oxidoreductase enzymes. Availability of this assembly and annotation provides a resource for further investigation of genomic organization in this species, and functional characterization of genes that may confer important phenotypic traits.

  13. Analysis of Neuronal Sequences Using Pairwise Biases

    Science.gov (United States)

    2015-08-27

    Structure of the Nervous System of the Nematode Caenorhabditis elegans”. In: Philosophical Transactions of the Royal Society B: Biological Sciences...oenalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR...data, and (2) it has no simple, realistic biological interpretation. In this chapter, we describe a new representation of sequences that addresses

  14. Evaluation of PGM sequencing platform using in bacterial genome de novo sequencing%ABIPGM测序平台用于细菌基因组denovo测序的评价

    Institute of Scientific and Technical Information of China (English)

    黄方亮

    2015-01-01

    为了探索加快细菌基因组研究的方法,利用ABI PGM测序平台测定了1株单细胞硫还原地杆菌的基因组序列。测序共获得1.4 Gbp 数据,平均读长为177 bp。通过多个拼接软件并采用合适的组装策略,得到一个完整细菌基因组3.55 Mbp和一条完整质粒序列110 kbp。测定基因组序列与参考基因组kn400序列的相似性达到94%,参考基因组91%的基因能在测定基因组中找到相似基因。通过本研究表明采用ABI PGM测序平台结合灵活的拼接策略可快速构建细菌基因组精细图谱,为进一步的功能注释及深入的信息分析提供准确的数据,大大加快研究进程。%In order to speed up bacterial genome exploration, we performed the genome sequencing of Geobacter sulfurreducens using PGM. Totally, 1. 4 Gbp raw data were obtained with an average read length of 177 bp. 2 contigs were assembled by multiple software calculations using appropriate assembly strategies. The size of whole obtained genome and plasmid was measured to be 3. 55 Mbp and 110 kbp, respectively. The sequenced genome identified 94% of reference genome strain KN400 and 91% genes of KN400 were tested to be orthologous in the sequenced genome. This study proved that the use of ABI PGM sequencing platform with splicing flexible strategy can rapidly build bacteria genome map. By providing accurate data for the functional annotation and in⁃depth information analysis, it will greatly accelerate research progress.

  15. Preliminary Results of De Novo Whole Genome Sequencing of the Siberian Larch (Larix sibirica Ledeb. and the Siberian Stone Pine (Pinus sibirica Du Tour

    Directory of Open Access Journals (Sweden)

    K. V. Krutovsky

    2014-08-01

    Full Text Available The Illumina HiSeq2000 DNA sequencing generated 2 906 977 265 high quality paired-end nucleotide sequences (reads and 576 Gbp for Siberian larch (Larix sibirica Ledeb. that corresponds to 48X coverage of the larch genome (12.03 Gbp, and 3 427 566 813 reads and 679 Gbp for Siberian stone pine (Pinus sibirica Du Tour that corresponds to 29X coverage of its genome (23.6 Gbp. These data are not enough to assemble and annotate whole genomes, but the obtained nucleotide sequences have allowed us to discover and develop effective highly polymorphic molecular genetic markers, such as microsatellite loci that are required for population genetic studies and identification of the timber origin. Sequence data can be used also to discover single nucleotide polymorphisms (SNPs. Genomic studies of Russian boreal forests and major phytopathogens associated with them will also allow us to identify biomarkers that can be used for solving important scientific and economic problems related to the conservation of forest genetic resources and breeding more resilient and fast growing trees with improved timber and resistance to diseases and adverse environmental factors.

  16. Cloning and sequence analysis of the partial sequence of the rbcL from Bryopsis hypnoides

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species,the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.

  17. CD20-negative de novo diffuse large B-cell lymphoma in HIV-negative patients: A matched case-control analysis in a single institution

    Directory of Open Access Journals (Sweden)

    Li Ya-Jun

    2012-05-01

    Full Text Available Abstract Background HIV-negative, CD20-negative de novo diffuse large B-cell lymphoma (DLBCL patients has rarely been reported. To elucidate the nature of this entity, we retrospectively reviewed the data of 1,456 consecutive de novo DLBCL patients who were treated at Sun Yat-Sen University Cancer Center between January 1999 and March 2011. Methods The pathologic characteristics of CD20-negative patients, clinical features, response to initial treatment, and outcomes of 28 patients with available clinical data (n = 21 were reviewed. Then, a matched case-control (1:3 analysis was performed to compare patients with CD20-negative and -positive DLBCL. Results The median age of the 28 CD20-negative DLBCL patients was 48 years, with a male-female ratio of 20:8. Seventeen of 22 (77.3% CD20-negative DLBCL cases were of the non-germinal centre B-cell (non-GCB subtype. High Ki67 expression (≥80%, an index of cell proliferation, was demonstrated in 17 of 24 (70.8% cases. Extranodal involvement (≥ 1 site was observed in 76.2% of the patients. Following initial therapy, 9 of 21 (42.9% cases achieved complete remission, 4 (19% achieved partial remission, 1 (4.8% had stable disease, and 7 (33.3% had disease progression. The median overall survival was 23 months. The 3-year progression-free survival (PFS and overall survival (OS rates were 30.5% and 35%, respectively. A matched case-control analysis showed that patients with CD20-negative and -positive DLBCL did not exhibit a statistically significant difference with respect to the main clinical characteristics (except extranodal involvement, whereas the patients with CD20-positive DLBCL had a better survival outcome with 3-year PFS (P = 0.008 and OS (P = 0.008 rates of 52% and 74.1%, respectively. Conclusions This study suggests that HIV-negative, CD20-negative de novo DLBCL patients have a higher proportion of non-GCB subtype, a higher proliferation index, more frequent extranodal involvement, a poorer

  18. Deep Sequencing Analysis of the Ixodes ricinus Haemocytome.

    Directory of Open Access Journals (Sweden)

    Michalis Kotsyfakis

    2015-05-01

    Full Text Available Ixodes ricinus is the main tick vector of the microbes that cause Lyme disease and tick-borne encephalitis in Europe. Pathogens transmitted by ticks have to overcome innate immunity barriers present in tick tissues, including midgut, salivary glands epithelia and the hemocoel. Molecularly, invertebrate immunity is initiated when pathogen recognition molecules trigger serum or cellular signalling cascades leading to the production of antimicrobials, pathogen opsonization and phagocytosis. We presently aimed at identifying hemocyte transcripts from semi-engorged female I. ricinus ticks by mass sequencing a hemocyte cDNA library and annotating immune-related transcripts based on their hemocyte abundance as well as their ubiquitous distribution.De novo assembly of 926,596 pyrosequence reads plus 49,328,982 Illumina reads (148 nt length from a hemocyte library, together with over 189 million Illumina reads from salivary gland and midgut libraries, generated 15,716 extracted coding sequences (CDS; these are displayed in an annotated hyperlinked spreadsheet format. Read mapping allowed the identification and annotation of tissue-enriched transcripts. A total of 327 transcripts were found significantly over expressed in the hemocyte libraries, including those coding for scavenger receptors, antimicrobial peptides, pathogen recognition proteins, proteases and protease inhibitors. Vitellogenin and lipid metabolism transcription enrichment suggests fat body components. We additionally annotated ubiquitously distributed transcripts associated with immune function, including immune-associated signal transduction proteins and transcription factors, including the STAT transcription factor.This is the first systems biology approach to describe the genes expressed in the haemocytes of this neglected disease vector. A total of 2,860 coding sequences were deposited to GenBank, increasing to 27,547 the number so far deposited by our previous transcriptome studies

  19. Protein comparative sequence analysis and computer modeling.

    Science.gov (United States)

    Hambly, Brett D; Oakley, Cecily E; Fajer, Piotr G

    2008-01-01

    A problem frequently encountered by the biological scientist is the identification of a previously unknown gene or protein sequence, where there are few or no clues as to the biochemical function, ligand specificity, gene regulation, protein-protein interactions, tissue specificity, cellular localization, developmental phase of activity, or biological role. Through the process of bioinformatics there are now many approaches for predicting answers to at least some of these questions, often then allowing the design of more insightful experiments to characterize more definitively the new protein.

  20. Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

    Directory of Open Access Journals (Sweden)

    Wadim L. Matochko

    2013-01-01

    Full Text Available Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N×1 frequency vector n=ni, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N×N matrix and a stochastic sampling operator (Sa. The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of Sa and use them to define the sequencing operator (Seq. Sequencing without any bias and errors is Seq=Sa IN, where IN is a N×N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (CEN, which describes elimination or statistically significant downsampling, of specific reads during the sequencing process.

  1. De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis and SNP markers development for rubber biosynthesis pathways.

    Directory of Open Access Journals (Sweden)

    Camila Campos Mantello

    Full Text Available Hevea brasiliensis (Willd. Ex Adr. Juss. Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr protein database returned 32,018 (63% positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG, gene ontology (GO, Kyoto Encyclopedia of Genes and Genomes (KEGG, and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs and single nucleotide polymorphisms (SNPs. In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA and 2-C-methyl-D-erythritol 4-phosphate (MEP pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

  2. De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis) and SNP markers development for rubber biosynthesis pathways.

    Science.gov (United States)

    Mantello, Camila Campos; Cardoso-Silva, Claudio Benicio; da Silva, Carla Cristina; de Souza, Livia Moura; Scaloppi Junior, Erivaldo José; de Souza Gonçalves, Paulo; Vicentini, Renato; de Souza, Anete Pereira

    2014-01-01

    Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

  3. De novo transcriptome sequence assembly and identification of AP2/ERF transcription factor related to abiotic stress in parsley (Petroselinum crispum).

    Science.gov (United States)

    Li, Meng-Yao; Tan, Hua-Wei; Wang, Feng; Jiang, Qian; Xu, Zhi-Sheng; Tian, Chang; Xiong, Ai-Sheng

    2014-01-01

    Parsley is an important biennial Apiaceae species that is widely cultivated as herb, spice, and vegetable. Previous studies on parsley principally focused on its physiological and biochemical properties, including phenolic compound and volatile oil contents. However, little is known about the molecular and genetic properties of parsley. In this study, 23,686,707 high-quality reads were obtained and assembled into 81,852 transcripts and 50,161 unigenes for the first time. Functional annotation showed that 30,516 unigenes had sequence similarity to known genes. In addition, 3,244 putative simple sequence repeats were detected in curly parsley. Finally, 1,569 of the identified unigenes belonged to 58 transcription factor families. Various abiotic stresses have a strong detrimental effect on the yield and quality of parsley. AP2/ERF transcription factors have important functions in plant development, hormonal regulation, and abiotic response. A total of 88 putative AP2/ERF factors were identified from the transcriptome sequence of parsley. Seven AP2/ERF transcription factors were selected in this study to analyze the expression profiles of parsley under different abiotic stresses. Our data provide a potentially valuable resource that can be used for intensive parsley research.

  4. De novo transcriptome sequence assembly and identification of AP2/ERF transcription factor related to abiotic stress in parsley (Petroselinum crispum.

    Directory of Open Access Journals (Sweden)

    Meng-Yao Li

    Full Text Available Parsley is an important biennial Apiaceae species that is widely cultivated as herb, spice, and vegetable. Previous studies on parsley principally focused on its physiological and biochemical properties, including phenolic compound and volatile oil contents. However, little is known about the molecular and genetic properties of parsley. In this study, 23,686,707 high-quality reads were obtained and assembled into 81,852 transcripts and 50,161 unigenes for the first time. Functional annotation showed that 30,516 unigenes had sequence similarity to known genes. In addition, 3,244 putative simple sequence repeats were detected in curly parsley. Finally, 1,569 of the identified unigenes belonged to 58 transcription factor families. Various abiotic stresses have a strong detrimental effect on the yield and quality of parsley. AP2/ERF transcription factors have important functions in plant development, hormonal regulation, and abiotic response. A total of 88 putative AP2/ERF factors were identified from the transcriptome sequence of parsley. Seven AP2/ERF transcription factors were selected in this study to analyze the expression profiles of parsley under different abiotic stresses. Our data provide a potentially valuable resource that can be used for intensive parsley research.

  5. Initial sequencing and analysis of the human genome.

    Science.gov (United States)

    Lander, E S; Linton, L M; Birren, B; Nusbaum, C; Zody, M C; Baldwin, J; Devon, K; Dewar, K; Doyle, M; FitzHugh, W; Funke, R; Gage, D; Harris, K; Heaford, A; Howland, J; Kann, L; Lehoczky, J; LeVine, R; McEwan, P; McKernan, K; Meldrim, J; Mesirov, J P; Miranda, C; Morris, W; Naylor, J; Raymond, C; Rosetti, M; Santos, R; Sheridan, A; Sougnez, C; Stange-Thomann, Y; Stojanovic, N; Subramanian, A; Wyman, D; Rogers, J; Sulston, J; Ainscough, R; Beck, S; Bentley, D; Burton, J; Clee, C; Carter, N; Coulson, A; Deadman, R; Deloukas, P; Dunham, A; Dunham, I; Durbin, R; French, L; Grafham, D; Gregory, S; Hubbard, T; Humphray, S; Hunt, A; Jones, M; Lloyd, C; McMurray, A; Matthews, L; Mercer, S; Milne, S; Mullikin, J C; Mungall, A; Plumb, R; Ross, M; Shownkeen, R; Sims, S; Waterston, R H; Wilson, R K; Hillier, L W; McPherson, J D; Marra, M A; Mardis, E R; Fulton, L A; Chinwalla, A T; Pepin, K H; Gish, W R; Chissoe, S L; Wendl, M C; Delehaunty, K D; Miner, T L; Delehaunty, A; Kramer, J B; Cook, L L; Fulton, R S; Johnson, D L; Minx, P J; Clifton, S W; Hawkins, T; Branscomb, E; Predki, P; Richardson, P; Wenning, S; Slezak, T; Doggett, N; Cheng, J F; Olsen, A; Lucas, S; Elkin, C; Uberbacher, E; Frazier, M; Gibbs, R A; Muzny, D M; Scherer, S E; Bouck, J B; Sodergren, E J; Worley, K C; Rives, C M; Gorrell, J H; Metzker, M L; Naylor, S L; Kucherlapati, R S; Nelson, D L; Weinstock, G M; Sakaki, Y; Fujiyama, A; Hattori, M; Yada, T; Toyoda, A; Itoh, T; Kawagoe, C; Watanabe, H; Totoki, Y; Taylor, T; Weissenbach, J; Heilig, R; Saurin, W; Artiguenave, F; Brottier, P; Bruls, T; Pelletier, E; Robert, C; Wincker, P; Smith, D R; Doucette-Stamm, L; Rubenfield, M; Weinstock, K; Lee, H M; Dubois, J; Rosenthal, A; Platzer, M; Nyakatura, G; Taudien, S; Rump, A; Yang, H; Yu, J; Wang, J; Huang, G; Gu, J; Hood, L; Rowen, L; Madan, A; Qin, S; Davis, R W; Federspiel, N A; Abola, A P; Proctor, M J; Myers, R M; Schmutz, J; Dickson, M; Grimwood, J; Cox, D R; Olson, M V; Kaul, R; Raymond, C; Shimizu, N; Kawasaki, K; Minoshima, S; Evans, G A; Athanasiou, M; Schultz, R; Roe, B A; Chen, F; Pan, H; Ramser, J; Lehrach, H; Reinhardt, R; McCombie, W R; de la Bastide, M; Dedhia, N; Blöcker, H; Hornischer, K; Nordsiek, G; Agarwala, R; Aravind, L; Bailey, J A; Bateman, A; Batzoglou, S; Birney, E; Bork, P; Brown, D G; Burge, C B; Cerutti, L; Chen, H C; Church, D; Clamp, M; Copley, R R; Doerks, T; Eddy, S R; Eichler, E E; Furey, T S; Galagan, J; Gilbert, J G; Harmon, C; Hayashizaki, Y; Haussler, D; Hermjakob, H; Hokamp, K; Jang, W; Johnson, L S; Jones, T A; Kasif, S; Kaspryzk, A; Kennedy, S; Kent, W J; Kitts, P; Koonin, E V; Korf, I; Kulp, D; Lancet, D; Lowe, T M; McLysaght, A; Mikkelsen, T; Moran, J V; Mulder, N; Pollara, V J; Ponting, C P; Schuler, G; Schultz, J; Slater, G; Smit, A F; Stupka, E; Szustakowki, J; Thierry-Mieg, D; Thierry-Mieg, J; Wagner, L; Wallis, J; Wheeler, R; Williams, A; Wolf, Y I; Wolfe, K H; Yang, S P; Yeh, R F; Collins, F; Guyer, M S; Peterson, J; Felsenfeld, A; Wetterstrand, K A; Patrinos, A; Morgan, M J; de Jong, P; Catanese, J J; Osoegawa, K; Shizuya, H; Choi, S; Chen, Y J; Szustakowki, J

    2001-02-15

    The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

  6. Genome-Wide Analysis of Secondary Metabolite Gene Clusters in Ophiostoma ulmi and Ophiostoma novo-ulmi Reveals a Fujikurin-Like Gene Cluster with a Putative Role in Infection

    Directory of Open Access Journals (Sweden)

    Nicolau Sbaraini

    2017-06-01

    Full Text Available The emergence of new microbial pathogens can result in destructive outbreaks, since their hosts have limited resistance and pathogens may be excessively aggressive. Described as the major ecological incident of the twentieth century, Dutch elm disease, caused by ascomycete fungi from the Ophiostoma genus, has caused a significant decline in elm tree populations (Ulmus sp. in North America and Europe. Genome sequencing of the two main causative agents of Dutch elm disease (Ophiostoma ulmi and Ophiostoma novo-ulmi, along with closely related species with different lifestyles, allows for unique comparisons to be made to identify how pathogens and virulence determinants have emerged. Among several established virulence determinants, secondary metabolites (SMs have been suggested to play significant roles during phytopathogen infection. Interestingly, the secondary metabolism of Dutch elm pathogens remains almost unexplored, and little is known about how SM biosynthetic genes are organized in these species. To better understand the metabolic potential of O. ulmi and O. novo-ulmi, we performed a deep survey and description of SM biosynthetic gene clusters (BGCs in these species and assessed their conservation among eight species from the Ophiostomataceae family. Among 19 identified BGCs, a fujikurin-like gene cluster (OpPKS8 was unique to Dutch elm pathogens. Phylogenetic analysis revealed that orthologs for this gene cluster are widespread among phytopathogens and plant-associated fungi, suggesting that OpPKS8 may have been horizontally acquired by the Ophiostoma genus. Moreover, the detailed identification of several BGCs paves the way for future in-depth research and supports the potential impact of secondary metabolism on Ophiostoma genus’ lifestyle.

  7. Facility Layout Based on Sequence Analysis: Design of Flowshops

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jin; WU Zhi-ming

    2009-01-01

    A computer-aided method to design a hybrid layout-tree-shape planar flowlines is presented. In new-type flowshop layout, the common machines shared by several flowlines could be located together in functional sections. The approach combines traditional cell formation techniques with sequence alignment algorithms. Firstly, a sequence analysis based cell formation procedure is adopted; then the operation sequences for parts are aligned to maximize machines adjacency in hyperedge representations; finally a tree-shape planar flowline will be obtained for each part family. With the help of a sample of operation sequences obtained from industry, this algorithm is illustrated.

  8. Mesoscopic Model for Free Energy Landscape Analysis of DNA sequences

    CERN Document Server

    Tapia-Rojo, R; Mazo, J J; Falo, F; 10.1103/PhysRevE.86.021908

    2012-01-01

    A mesoscopic model which allows us to identify and quantify the strength of binding sites in DNA sequences is proposed. The model is based on the Peyrard-Bishop-Dauxois model for the DNA chain coupled to a Brownian particle which explores the sequence interacting more importantly with open base pairs of the DNA chain. We apply the model to promoter sequences of different organisms. The free energy landscape obtained for these promoters shows a complex structure that is strongly connected to their biological behavior. The analysis method used is able to quantify free energy differences of sites within genome sequences.

  9. Real analysis via sequences and series

    CERN Document Server

    Little, Charles H C; van Brunt, Bruce

    2015-01-01

    This text gives a rigorous treatment of the foundations of calculus. In contrast to more traditional approaches, infinite sequences and series are placed at the forefront. The approach taken has not only the merit of simplicity, but students are well placed to understand and appreciate more sophisticated concepts in advanced mathematics. The authors mitigate potential difficulties in mastering the material by motivating  definitions, results, and proofs. Simple examples  are provided to  illustrate new material and exercises are included at the end of most sections. Noteworthy topics include: an extensive discussion of convergence tests for infinite series, Wallis’s formula and Stirling’s formula, proofs of the irrationality of π and e, and a treatment of Newton’s method as a special instance of finding fixed points of iterated functions.

  10. De novo genome assembly and annotation of Australia's largest freshwater fish, the Murray cod (Maccullochella peelii), from Illumina and Nanopore sequencing read.

    Science.gov (United States)

    Austin, Christopher M; Tan, Mun Hua; Harrisson, Katherine A; Lee, Yin Peng; Croft, Laurence J; Sunnucks, Paul; Pavlova, Alexandra; Gan, Han Ming

    2017-08-01

    One of the most iconic Australian fish is the Murray cod, Maccullochella peelii (Mitchell 1838), a freshwater species that can grow to ∼1.8 metres in length and live to age ≥48 years. The Murray cod is of a conservation concern as a result of strong population contractions, but it is also popular for recreational fishing and is of growing aquaculture interest. In this study, we report the whole genome sequence of the Murray cod to support ongoing population genetics, conservation, and management research, as well as to better understand the evolutionary ecology and history of the species. A draft Murray cod genome of 633 Mbp (N50 = 109 974bp; BUSCO and CEGMA completeness of 94.2% and 91.9%, respectively) with an estimated 148 Mbp of putative repetitive sequences was assembled from the combined sequencing data of 2 fish individuals with an identical maternal lineage; 47.2 Gb of Illumina HiSeq data and 804 Mb of Nanopore data were generated from the first individual while 23.2 Gb of Illumina MiSeq data were generated from the second individual. The inclusion of Nanopore reads for scaffolding followed by subsequent gap-closing using Illumina data led to a 29% reduction in the number of scaffolds and a 55% and 54% increase in the scaffold and contig N50, respectively. We also report the first transcriptome of Murray cod that was subsequently used to annotate the Murray cod genome, leading to the identification of 26 539 protein-coding genes. We present the whole genome of the Murray cod and anticipate this will be a catalyst for a range of genetic, genomic, and phylogenetic studies of the Murray cod and more generally other fish species of the Percichthydae family. © The Authors 2017. Published by Oxford University Press.

  11. Editorial: Special Issue on Algorithms for Sequence Analysis and Storage

    Directory of Open Access Journals (Sweden)

    Veli Mäkinen

    2014-03-01

    Full Text Available This special issue of Algorithms is dedicated to approaches to biological sequence analysis that have algorithmic novelty and potential for fundamental impact in methods used for genome research.

  12. De novo assembly of a haplotype-resolved human genome.

    Science.gov (United States)

    Cao, Hongzhi; Wu, Honglong; Luo, Ruibang; Huang, Shujia; Sun, Yuhui; Tong, Xin; Xie, Yinlong; Liu, Binghang; Yang, Hailong; Zheng, Hancheng; Li, Jian; Li, Bo; Wang, Yu; Yang, Fang; Sun, Peng; Liu, Siyang; Gao, Peng; Huang, Haodong; Sun, Jing; Chen, Dan; He, Guangzhu; Huang, Weihua; Huang, Zheng; Li, Yue; Tellier, Laurent C A M; Liu, Xiao; Feng, Qiang; Xu, Xun; Zhang, Xiuqing; Bolund, Lars; Krogh, Anders; Kristiansen, Karsten; Drmanac, Radoje; Drmanac, Snezana; Nielsen, Rasmus; Li, Songgang; Wang, Jian; Yang, Huanming; Li, Yingrui; Wong, Gane Ka-Shu; Wang, Jun

    2015-06-01

    The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

  13. De novo assembly of a haplotype-resolved human genome

    DEFF Research Database (Denmark)

    Cao, Hongzhi; Wu, Honglong; Luo, Ruibang

    2015-01-01

    The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome...... of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should...... shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb...

  14. [Automatic analysis pipeline of next-generation sequencing data].

    Science.gov (United States)

    Wenke, Li; Fengyu, Li; Siyao, Zhang; Bin, Cai; Na, Zheng; Yu, Nie; Dao, Zhou; Qian, Zhao

    2014-06-01

    The development of next-generation sequencing has generated high demand for data processing and analysis. Although there are a lot of software for analyzing next-generation sequencing data, most of them are designed for one specific function (e.g., alignment, variant calling or annotation). Therefore, it is necessary to combine them together for data analysis and to generate interpretable results for biologists. This study designed a pipeline to process Illumina sequencing data based on Perl programming language and SGE system. The pipeline takes original sequence data (fastq format) as input, calls the standard data processing software (e.g., BWA, Samtools, GATK, and Annovar), and finally outputs a list of annotated variants that researchers can further analyze. The pipeline simplifies the manual operation and improves the efficiency by automatization and parallel computation. Users can easily run the pipeline by editing the configuration file or clicking the graphical interface. Our work will facilitate the research projects using the sequencing technology.

  15. The Spectrum Sequences of Periodic Frame Multiresolution Analysis

    Institute of Scientific and Technical Information of China (English)

    Yun Zhang LI; Qiao Fang LIAN

    2009-01-01

    The periodic multiresolution analysis (PMRA) and the periodic frame multiresolution analysis (PFMRA) provide a general recipe for the construction of periodic wavelets and periodic wavelet frames,respectively. This paper addresses PFMRAs by the introduction of the notion of spec-trum sequence. In terms of spectrum sequences,the scaling function sequences generating a normalized PFMRA are characterized; a characterization of the spectrum sequences of PFMRAs is obtained,which provides a method to construct PFMRAs since its proof is constructive; a necessary and sufficient con-dition for a PFMRA to admit a single wavelet frame sequence is obtained; a necessary and sufficient condition for a PFMRA to be contained in a given PMRA is also obtained. What is more,it is proved that an arbitrary PFMRA must be contained in some PMRA. In the meanwhile,some examples are provided to illustrate the general theory.

  16. De novo Transcriptome Generation and Annotation for Two Korean Endemic Land Snails, Aegista chejuensis and Aegista quelpartensis, Using Illumina Paired-End Sequencing Technology

    Directory of Open Access Journals (Sweden)

    Se Won Kang

    2016-03-01

    Full Text Available Aegista chejuensis and Aegista quelpartensis (Family-Bradybaenidae are endemic to Korea, and are considered vulnerable due to declines in their population. The limited genetic resources for these species restricts the ability to prioritize conservation efforts. We sequenced the transcriptomes of these species using Illumina paired-end technology. Approximately 257 and 240 million reads were obtained and assembled into 198,531 and 230,497 unigenes for A. chejuensis and A. quelpartensis, respectively. The average and N50 unigene lengths were 735.4 and 1073 bp, respectively, for A. chejuensis, and 705.6 and 1001 bp, respectively, for A. quelpartensis. In total, 68,484 (34.5% and 77,745 (33.73% unigenes for A. chejuensis and A. quelpartensis, respectively, were annotated to databases. Gene Ontology terms were assigned to 23,778 (11.98% and 26,396 (11.45 unigenes, for A. chejuensis and A. quelpartensis, respectively, while 5050 and 5838 unigenes were mapped to 117 and 124 pathways in the Kyoto Encyclopedia of Genes and Genomes database. In addition, we identified and annotated 9542 and 10,395 putative simple sequence repeats (SSRs in unigenes from A. chejuensis and A. quelpartensis, respectively. We designed a list of PCR primers flanking the putative SSR regions. These microsatellites may be utilized for future phylogenetics and conservation initiatives.

  17. De-novo RNA sequencing and metabolite profiling to identify genes involved in anthocyanin biosynthesis in Korean black raspberry (Rubus coreanus Miquel).

    Science.gov (United States)

    Hyun, Tae Kyung; Lee, Sarah; Rim, Yeonggil; Kumar, Ritesh; Han, Xiao; Lee, Sang Yeol; Lee, Choong Hwan; Kim, Jae-Yean

    2014-01-01

    The Korean black raspberry (Rubus coreanus Miquel, KB) on ripening is usually consumed as fresh fruit, whereas the unripe KB has been widely used as a source of traditional herbal medicine. Such a stage specific utilization of KB has been assumed due to the changing metabolite profile during fruit ripening process, but so far molecular and biochemical changes during its fruit maturation are poorly understood. To analyze biochemical changes during fruit ripening process at molecular level, firstly, we have sequenced, assembled, and annotated the transcriptome of KB fruits. Over 4.86 Gb of normalized cDNA prepared from fruits was sequenced using Illumina HiSeq™ 2000, and assembled into 43,723 unigenes. Secondly, we have reported that alterations in anthocyanins and proanthocyanidins are the major factors facilitating variations in these stages of fruits. In addition, up-regulation of F3'H1, DFR4 and LDOX1 resulted in the accumulation of cyanidin derivatives during the ripening process of KB, indicating the positive relationship between the expression of anthocyanin biosynthetic genes and the anthocyanin accumulation. Furthermore, the ability of RcMCHI2 (R. coreanus Miquel chalcone flavanone isomerase 2) gene to complement Arabidopsis transparent testa 5 mutant supported the feasibility of our transcriptome library to provide the gene resources for improving plant nutrition and pigmentation. Taken together, these datasets obtained from transcriptome library and metabolic profiling would be helpful to define the gene-metabolite relationships in this non-model plant.

  18. De-novo RNA sequencing and metabolite profiling to identify genes involved in anthocyanin biosynthesis in Korean black raspberry (Rubus coreanus Miquel.

    Directory of Open Access Journals (Sweden)

    Tae Kyung Hyun

    Full Text Available The Korean black raspberry (Rubus coreanus Miquel, KB on ripening is usually consumed as fresh fruit, whereas the unripe KB has been widely used as a source of traditional herbal medicine. Such a stage specific utilization of KB has been assumed due to the changing metabolite profile during fruit ripening process, but so far molecular and biochemical changes during its fruit maturation are poorly understood. To analyze biochemical changes during fruit ripening process at molecular level, firstly, we have sequenced, assembled, and annotated the transcriptome of KB fruits. Over 4.86 Gb of normalized cDNA prepared from fruits was sequenced using Illumina HiSeq™ 2000, and assembled into 43,723 unigenes. Secondly, we have reported that alterations in anthocyanins and proanthocyanidins are the major factors facilitating variations in these stages of fruits. In addition, up-regulation of F3'H1, DFR4 and LDOX1 resulted in the accumulation of cyanidin derivatives during the ripening process of KB, indicating the positive relationship between the expression of anthocyanin biosynthetic genes and the anthocyanin accumulation. Furthermore, the ability of RcMCHI2 (R. coreanus Miquel chalcone flavanone isomerase 2 gene to complement Arabidopsis transparent testa 5 mutant supported the feasibility of our transcriptome library to provide the gene resources for improving plant nutrition and pigmentation. Taken together, these datasets obtained from transcriptome library and metabolic profiling would be helpful to define the gene-metabolite relationships in this non-model plant.

  19. Sequencing and Analysis of Neanderthal Genomic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Noonan, James P.; Coop, Graham; Kudaravalli, Sridhar; Smith,Doug; Krause, Johannes; Alessi, Joe; Chen, Feng; Platt, Darren; Paabo,Svante; Pritchard, Jonathan K.; Rubin, Edward M.

    2006-06-13

    Recovery and analysis of multiple Neanderthal autosomalsequences using a metagenomic approach reveals that modern humans andNeanderthals split ~;400,000 years ago, without significant evidence ofsubsequent admixture.

  20. DNA methylation analysis of human myoblasts during in vitro myogenic differentiation: de novo methylation of promoters of muscle-related genes and its involvement in transcriptional down-regulation.

    Science.gov (United States)

    Miyata, Kohei; Miyata, Tomoko; Nakabayashi, Kazuhiko; Okamura, Kohji; Naito, Masashi; Kawai, Tomoko; Takada, Shuji; Kato, Kiyoko; Miyamoto, Shingo; Hata, Kenichiro; Asahara, Hiroshi

    2015-01-15

    Although DNA methylation is considered to play an important role during myogenic differentiation, chronological alterations in DNA methylation and gene expression patterns in this process have been poorly understood. Using the Infinium HumanMethylation450 BeadChip array, we obtained a chronological profile of the genome-wide DNA methylation status in a human myoblast differentiation model, where myoblasts were cultured in low-serum medium to stimulate myogenic differentiation. As the differentiation of the myoblasts proceeded, their global DNA methylation level increased and their methylation patterns became more distinct from those of mesenchymal stem cells. Gene ontology analysis revealed that genes whose promoter region was hypermethylated upon myoblast differentiation were highly significantly enriched with muscle-related terms such as 'muscle contraction' and 'muscle system process'. Sequence motif analysis identified 8-bp motifs somewhat similar to the binding motifs of ID4 and ZNF238 to be most significantly enriched in hypermethylated promoter regions. ID4 and ZNF238 have been shown to be critical transcriptional regulators of muscle-related genes during myogenic differentiation. An integrated analysis of DNA methylation and gene expression profiles revealed that de novo DNA methylation of non-CpG island (CGI) promoters was more often associated with transcriptional down-regulation than that of CGI promoters. These results strongly suggest the existence of an epigenetic mechanism in which DNA methylation modulates the functions of key transcriptional factors to coordinately regulate muscle-related genes during myogenic differentiation.

  1. Inference of splicing regulatory activities by sequence neighborhood analysis.

    Directory of Open Access Journals (Sweden)

    Michael B Stadler

    2006-11-01

    Full Text Available Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE and silencer (ESS hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences.

  2. Tools for integrated sequence-structure analysis with UCSF Chimera

    Directory of Open Access Journals (Sweden)

    Huang Conrad C

    2006-07-01

    Full Text Available Abstract Background Comparing related structures and viewing the structures in the context of sequence alignments are important tasks in protein structure-function research. While many programs exist for individual aspects of such work, there is a need for interactive visualization tools that: (a provide a deep integration of sequence and structure, far beyond mapping where a sequence region falls in the structure and vice versa; (b facilitate changing data of one type based on the other (for example, using only sequence-conserved residues to match structures, or adjusting a sequence alignment based on spatial fit; (c can be used with a researcher's own data, including arbitrary sequence alignments and annotations, closely or distantly related sets of proteins, etc.; and (d interoperate with each other and with a full complement of molecular graphics features. We describe enhancements to UCSF Chimera to achieve these goals. Results The molecular graphics program UCSF Chimera includes a suite of tools for interactive analyses of sequences and structures. Structures automatically associate with sequences in imported alignments, allowing many kinds of crosstalk. A novel method is provided to superimpose structures in the absence of a pre-existing sequence alignment. The method uses both sequence and secondary structure, and can match even structures with very low sequence identity. Another tool constructs structure-based sequence alignments from superpositions of two or more proteins. Chimera is designed to be extensible, and mechanisms for incorporating user-specific data without Chimera code development are also provided. Conclusion The tools described here apply to many problems involving comparison and analysis of protein structures and their sequences. Chimera includes complete documentation and is intended for use by a wide range of scientists, not just those in the computational disciplines. UCSF Chimera is free for non-commercial use and is

  3. De novo foliar transcriptome of Chenopodium amaranticolor and analysis of its gene expression during virus-induced hypersensitive response.

    Directory of Open Access Journals (Sweden)

    Yongqiang Zhang

    Full Text Available BACKGROUND: The hypersensitive response (HR system of Chenopodium spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for Chenopodium, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of C. amaranticolor, a Chenopodium species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance. METHODOLOGY AND PRINCIPAL FINDINGS: Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons. BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10(-5. Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by Tobacco mosaic virus and Cucumber mosaic virus by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized. CONCLUSIONS: To our knowledge, this study is the first description of the genetic makeup of C. amaranticolor, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further

  4. De Novo Assembly and Transcriptome Analysis of Wheat with Male Sterility Induced by the Chemical Hybridizing Agent SQ-1.

    Directory of Open Access Journals (Sweden)

    Qidi Zhu

    Full Text Available Wheat (Triticum aestivum L., one of the world's most important food crops, is a strictly autogamous (self-pollinating species with exclusively perfect flowers. Male sterility induced by chemical hybridizing agents has increasingly attracted attention as a tool for hybrid seed production in wheat; however, the molecular mechanisms of male sterility induced by the agent SQ-1 remain poorly understood due to limited whole transcriptome data. Therefore, a comparative analysis of wheat anther transcriptomes for male fertile wheat and SQ-1-induced male sterile wheat was carried out using next-generation sequencing technology. In all, 42,634,123 sequence reads were generated and were assembled into 82,356 high-quality unigenes with an average length of 724 bp. Of these, 1,088 unigenes were significantly differentially expressed in the fertile and sterile wheat anthers, including 643 up-regulated unigenes and 445 down-regulated unigenes. The differentially expressed unigenes with functional annotations were mapped onto 60 pathways using the Kyoto Encyclopedia of Genes and Genomes database. They were mainly involved in coding for the components of ribosomes, photosynthesis, respiration, purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, RNA transport and signal transduction, reactive oxygen species metabolism, mRNA surveillance pathways, protein processing in the endoplasmic reticulum, protein export, and ubiquitin-mediated proteolysis. This study is the first to provide a systematic overview comparing wheat anther transcriptomes of male fertile wheat with those of SQ-1-induced male sterile wheat and is a valuable source of data for future research in SQ-1-induced wheat male sterility.

  5. DSAP: deep-sequencing small RNA analysis pipeline.

    Science.gov (United States)

    Huang, Po-Jung; Liu, Yi-Chung; Lee, Chi-Ching; Lin, Wei-Chen; Gan, Richie Ruei-Chi; Lyu, Ping-Chiang; Tang, Petrus

    2010-07-01

    DSAP is an automated multiple-task web service designed to provide a total solution to analyzing deep-sequencing small RNA datasets generated by next-generation sequencing technology. DSAP uses a tab-delimited file as an input format, which holds the unique sequence reads (tags) and their corresponding number of copies generated by the Solexa sequencing platform. The input data will go through four analysis steps in DSAP: (i) cleanup: removal of adaptors and poly-A/T/C/G/N nucleotides; (ii) clustering: grouping of cleaned sequence tags into unique sequence clusters; (iii) non-coding RNA (ncRNA) matching: sequence homology mapping against a transcribed sequence library from the ncRNA database Rfam (http://rfam.sanger.ac.uk/); and (iv) known miRNA matching: detection of known miRNAs in miRBase (http://www.mirbase.org/) based on sequence homology. The expression levels corresponding to matched ncRNAs and miRNAs are summarized in multi-color clickable bar charts linked to external databases. DSAP is also capable of displaying miRNA expression levels from different jobs using a log(2)-scaled color matrix. Furthermore, a cross-species comparative function is also provided to show the distribution of identified miRNAs in different species as deposited in miRBase. DSAP is available at http://dsap.cgu.edu.tw.

  6. Comprehensive analysis of cooperative gene mutations between class I and class II in de novo acute myeloid leukemia.

    Science.gov (United States)

    Ishikawa, Yuichi; Kiyoi, Hitoshi; Tsujimura, Akane; Miyawaki, Shuichi; Miyazaki, Yasushi; Kuriyama, Kazutaka; Tomonaga, Masao; Naoe, Tomoki

    2009-08-01

    Acute myeloid leukemia (AML) has been thought to be the consequence of two broad complementation classes of mutations: class I and class II. However, overlap-mutations between them or within the same class and the position of TP53 mutation are not fully analyzed. We comprehensively analyzed the FLT3, cKIT, N-RAS, C/EBPA, AML1, MLL, NPM1, and TP53 mutations in 144 newly diagnosed de novo AML. We found 103 of 165 identified mutations were overlapped with other mutations, and most overlap-mutations consisted of class I and class II mutations. Although overlap-mutations within the same class were found in seven patients, five of them additionally had the other class mutation. These results suggest that most overlap-mutations within the same class might be the consequence of acquiring an additional mutation after the completion both of class I and class II mutations. However, mutated genes overlapped with the same class were limited in N-RAS, TP53, MLL-PTD, and NPM1, suggesting the possibility that these irregular overlap-mutations might cooperatively participate in the development of AML. Notably, TP53 mutation was overlapped with both class I and class II mutations, and associated with morphologic multilineage dysplasia and complex karyotype. The genotype consisting of complex karyotype and TP53 mutation was an unfavorable prognostic factor in entire AML patients, indicating this genotype generates a disease entity in de novo AML. These results collectively suggest that TP53 mutation might be a functionally distinguishable class of mutation.

  7. Event Sequence Analysis using Self Organizing Map

    OpenAIRE

    2012-01-01

    In today’s world we have abundance of data and scarcity of Knowledge data mining field emerged as the fit of thetool to the problem. With the advent of internet technology and the exponential growth in the technology behind theworld wide web, the concept of web mining and found a place for itself and emerged as a separate field of research.Web mining involves a wide range of applications that aim at discovering and extracting hidden information in datastored on the Web. Web log analysis is an...

  8. Mammalian comparative sequence analysis of the Agrp locus.

    Directory of Open Access Journals (Sweden)

    Christopher B Kaelin

    Full Text Available Agouti-related protein encodes a neuropeptide that stimulates food intake. Agrp expression in the brain is restricted to neurons in the arcuate nucleus of the hypothalamus and is elevated by states of negative energy balance. The molecular mechanisms underlying Agrp regulation, however, remain poorly defined. Using a combination of transgenic and comparative sequence analysis, we have previously identified a 760 bp conserved region upstream of Agrp which contains STAT binding elements that participate in Agrp transcriptional regulation. In this study, we attempt to improve the specificity for detecting conserved elements in this region by comparing genomic sequences from 10 mammalian species. Our analysis reveals a symmetrical organization of conserved sequences upstream of Agrp, which cluster into two inverted repeat elements. Conserved sequences within these elements suggest a role for homeodomain proteins in the regulation of Agrp and provide additional targets for functional evaluation.

  9. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  10. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  11. Applications of recursive segmentation to the analysis of DNA sequences.

    Science.gov (United States)

    Li, Wentian; Bernaola-Galván, Pedro; Haghighi, Fatameh; Grosse, Ivo

    2002-07-01

    Recursive segmentation is a procedure that partitions a DNA sequence into domains with a homogeneous composition of the four nucleotides A, C, G and T. This procedure can also be applied to any sequence converted from a DNA sequence, such as to a binary strong(G + C)/weak(A + T) sequence, to a binary sequence indicating the presence or absence of the dinucleotide CpG, or to a sequence indicating both the base and the codon position information. We apply various conversion schemes in order to address the following five DNA sequence analysis problems: isochore mapping, CpG island detection, locating the origin and terminus of replication in bacterial genomes, finding complex repeats in telomere sequences, and delineating coding and noncoding regions. We find that the recursive segmentation procedure can successfully detect isochore borders, CpG islands, and the origin and terminus of replication, but it needs improvement for detecting complex repeats as well as borders between coding and noncoding regions.

  12. Statistical design and analysis of RNA sequencing data.

    Science.gov (United States)

    Auer, Paul L; Doerge, R W

    2010-06-01

    Next-generation sequencing technologies are quickly becoming the preferred approach for characterizing and quantifying entire genomes. Even though data produced from these technologies are proving to be the most informative of any thus far, very little attention has been paid to fundamental design aspects of data collection and analysis, namely sampling, randomization, replication, and blocking. We discuss these concepts in an RNA sequencing framework. Using simulations we demonstrate the benefits of collecting replicated RNA sequencing data according to well known statistical designs that partition the sources of biological and technical variation. Examples of these designs and their corresponding models are presented with the goal of testing differential expression.

  13. An optimum analysis sequence for environmental gamma-ray spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    De la Torre, F.; Rios M, C.; Ruvalcaba A, M. G.; Mireles G, F.; Saucedo A, S.; Davila R, I.; Pinedo, J. L., E-mail: fta777@hotmail.co [Universidad Autonoma de Zacatecas, Centro Regional de Estudis Nucleares, Calle Cipres No. 10, Fracc. La Penuela, 98068 Zacatecas (Mexico)

    2010-10-15

    This work aims to obtain an optimum analysis sequence for environmental gamma-ray spectroscopy by means of Genie 2000 (Canberra). Twenty different analysis sequences were customized using different peak area percentages and different algorithms for: 1) peak finding, and 2) peak area determination, and with or without the use of a library -based on evaluated nuclear data- of common gamma-ray emitters in environmental samples. The use of an optimum analysis sequence with certified nuclear information avoids the problems originated by the significant variations in out-of-date nuclear parameters of commercial software libraries. Interference-free gamma ray energies with absolute emission probabilities greater than 3.75% were included in the customized library. The gamma-ray spectroscopy system (based on a Ge Re-3522 Canberra detector) was calibrated both in energy and shape by means of the IAEA-2002 reference spectra for software intercomparison. To test the performance of the analysis sequences, the IAEA-2002 reference spectrum was used. The z-score and the reduced {chi}{sup 2} criteria were used to determine the optimum analysis sequence. The results show an appreciable variation in the peak area determinations and their corresponding uncertainties. Particularly, the combination of second derivative peak locate with simple peak area integration algorithms provides the greater accuracy. Lower accuracy comes from the combination of library directed peak locate algorithm and Genie's Gamma-M peak area determination. (Author)

  14. Sequence and analysis of the genome of the pathogenic yeast Candida orthopsilosis.

    Science.gov (United States)

    Riccombeni, Alessandro; Vidanes, Genevieve; Proux-Wéra, Estelle; Wolfe, Kenneth H; Butler, Geraldine

    2012-01-01

    Candida orthopsilosis is closely related to the fungal pathogen Candida parapsilosis. However, whereas C. parapsilosis is a major cause of disease in immunosuppressed individuals and in premature neonates, C. orthopsilosis is more rarely associated with infection. We sequenced the C. orthopsilosis genome to facilitate the identification of genes associated with virulence. Here, we report the de novo assembly and annotation of the genome of a Type 2 isolate of C. orthopsilosis. The sequence was obtained by combining data from next generation sequencing (454 Life Sciences and Illumina) with paired-end Sanger reads from a fosmid library. The final assembly contains 12.6 Mb on 8 chromosomes. The genome was annotated using an automated pipeline based on comparative analysis of genomes of Candida species, together with manual identification of introns. We identified 5700 protein-coding genes in C. orthopsilosis, of which 5570 have an ortholog in C. parapsilosis. The time of divergence between C. orthopsilosis and C. parapsilosis is estimated to be twice as great as that between Candida albicans and Candida dubliniensis. There has been an expansion of the Hyr/Iff family of cell wall genes and the JEN family of monocarboxylic transporters in C. parapsilosis relative to C. orthopsilosis. We identified one gene from a Maltose/Galactoside O-acetyltransferase family that originated by horizontal gene transfer from a bacterium to the common ancestor of C. orthopsilosis and C. parapsilosis. We report that TFB3, a component of the general transcription factor TFIIH, undergoes alternative splicing by intron retention in multiple Candida species. We also show that an intein in the vacuolar ATPase gene VMA1 is present in C. orthopsilosis but not C. parapsilosis, and has a patchy distribution in Candida species. Our results suggest that the difference in virulence between C. parapsilosis and C. orthopsilosis may be associated with expansion of gene families.

  15. Sequence and Analysis of the Genome of the Pathogenic Yeast Candida orthopsilosis

    Science.gov (United States)

    Riccombeni, Alessandro; Vidanes, Genevieve; Proux-Wéra, Estelle; Wolfe, Kenneth H.; Butler, Geraldine

    2012-01-01

    Candida orthopsilosis is closely related to the fungal pathogen Candida parapsilosis. However, whereas C. parapsilosis is a major cause of disease in immunosuppressed individuals and in premature neonates, C. orthopsilosis is more rarely associated with infection. We sequenced the C. orthopsilosis genome to facilitate the identification of genes associated with virulence. Here, we report the de novo assembly and annotation of the genome of a Type 2 isolate of C. orthopsilosis. The sequence was obtained by combining data from next generation sequencing (454 Life Sciences and Illumina) with paired-end Sanger reads from a fosmid library. The final assembly contains 12.6 Mb on 8 chromosomes. The genome was annotated using an automated pipeline based on comparative analysis of genomes of Candida species, together with manual identification of introns. We identified 5700 protein-coding genes in C. orthopsilosis, of which 5570 have an ortholog in C. parapsilosis. The time of divergence between C. orthopsilosis and C. parapsilosis is estimated to be twice as great as that between Candida albicans and Candida dubliniensis. There has been an expansion of the Hyr/Iff family of cell wall genes and the JEN family of monocarboxylic transporters in C. parapsilosis relative to C. orthopsilosis. We identified one gene from a Maltose/Galactoside O-acetyltransferase family that originated by horizontal gene transfer from a bacterium to the common ancestor of C. orthopsilosis and C. parapsilosis. We report that TFB3, a component of the general transcription factor TFIIH, undergoes alternative splicing by intron retention in multiple Candida species. We also show that an intein in the vacuolar ATPase gene VMA1 is present in C. orthopsilosis but not C. parapsilosis, and has a patchy distribution in Candida species. Our results suggest that the difference in virulence between C. parapsilosis and C. orthopsilosis may be associated with expansion of gene families. PMID:22563396

  16. Sequence and analysis of the genome of the pathogenic yeast Candida orthopsilosis.

    Directory of Open Access Journals (Sweden)

    Alessandro Riccombeni

    Full Text Available Candida orthopsilosis is closely related to the fungal pathogen Candida parapsilosis. However, whereas C. parapsilosis is a major cause of disease in immunosuppressed individuals and in premature neonates, C. orthopsilosis is more rarely associated with infection. We sequenced the C. orthopsilosis genome to facilitate the identification of genes associated with virulence. Here, we report the de novo assembly and annotation of the genome of a Type 2 isolate of C. orthopsilosis. The sequence was obtained by combining data from next generation sequencing (454 Life Sciences and Illumina with paired-end Sanger reads from a fosmid library. The final assembly contains 12.6 Mb on 8 chromosomes. The genome was annotated using an automated pipeline based on comparative analysis of genomes of Candida species, together with manual identification of introns. We identified 5700 protein-coding genes in C. orthopsilosis, of which 5570 have an ortholog in C. parapsilosis. The time of divergence between C. orthopsilosis and C. parapsilosis is estimated to be twice as great as that between Candida albicans and Candida dubliniensis. There has been an expansion of the Hyr/Iff family of cell wall genes and the JEN family of monocarboxylic transporters in C. parapsilosis relative to C. orthopsilosis. We identified one gene from a Maltose/Galactoside O-acetyltransferase family that originated by horizontal gene transfer from a bacterium to the common ancestor of C. orthopsilosis and C. parapsilosis. We report that TFB3, a component of the general transcription factor TFIIH, undergoes alternative splicing by intron retention in multiple Candida species. We also show that an intein in the vacuolar ATPase gene VMA1 is present in C. orthopsilosis but not C. parapsilosis, and has a patchy distribution in Candida species. Our results suggest that the difference in virulence between C. parapsilosis and C. orthopsilosis may be associated with expansion of gene families.

  17. Whole genome sequence analysis of the arctic-lineage strain responsible for distemper in Italian wolves and dogs through a fast and robust next generation sequencing protocol.

    Science.gov (United States)

    Marcacci, Maurilia; Ancora, Massimo; Mangone, Iolanda; Teodori, Liana; Di Sabatino, Daria; De Massis, Fabrizio; Camma', Cesare; Savini, Giovanni; Lorusso, Alessio

    2014-06-01

    Dynamic surveillance and characterization of canine distemper virus (CDV) circulating strains are essential against possible vaccine breakthroughs events. This study describes the setup of a fast and robust next-generation sequencing (NGS) Ion PGM™ protocol that was used to obtain the complete genome sequence of a CDV isolate (CDV2784/2013). CDV2784/2013 is the prototype of CDV strains responsible for severe clinical distemper in dogs and wolves in Italy during 2013. CDV2784/2013 was isolated on cell culture and total RNA was used for NGS sample preparation. A total of 112.3 Mb of reads were assembled de novo using MIRA version 4.0rc4, which yielded a total number of 403 contigs with 12.1% coverage. The whole genome (15,690 bp) was recovered successfully and compared to those of existing CDV whole genomes. CDV2784/2013 was shown to have 92% nt identity with the Onderstepoort vaccine strain. This study describes for the first time a fast and robust Ion PGM™ platform-based whole genome amplification protocol for non-segmented negative stranded RNA viruses starting from total cell-purified RNA. Additionally, this is the first study reporting the whole genome analysis of an Arctic lineage strain that is known to circulate widely in Europe, Asia and USA.

  18. Deep sequencing analysis of phage libraries using Illumina platform.

    Science.gov (United States)

    Matochko, Wadim L; Chu, Kiki; Jin, Bingjie; Lee, Sam W; Whitesides, George M; Derda, Ratmir

    2012-09-01

    This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve

  19. High Throughput Plasmid Sequencing with Illumina and CLC Bio (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    Energy Technology Data Exchange (ETDEWEB)

    Athavale, Ajay [Monsanto

    2012-06-01

    Ajay Athavale (Monsanto) presents "High Throughput Plasmid Sequencing with Illumina and CLC Bio" at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  20. Frequency and Complexity of De Novo Structural Mutation in Autism

    Science.gov (United States)

    Brandler, William M.; Antaki, Danny; Gujral, Madhusudan; Noor, Amina; Rosanio, Gabriel; Chapman, Timothy R.; Barrera, Daniel J.; Lin, Guan Ning; Malhotra, Dheeraj; Watts, Amanda C.; Wong, Lawrence C.; Estabillo, Jasper A.; Gadomski, Therese E.; Hong, Oanh; Fajardo, Karin V. Fuentes; Bhandari, Abhishek; Owen, Renius; Baughn, Michael; Yuan, Jeffrey; Solomon, Terry; Moyzis, Alexandra G.; Maile, Michelle S.; Sanders, Stephan J.; Reiner, Gail E.; Vaux, Keith K.; Strom, Charles M.; Zhang, Kang; Muotri, Alysson R.; Akshoomoff, Natacha; Leal, Suzanne M.; Pierce, Karen; Courchesne, Eric; Iakoucheva, Lilia M.; Corsello, Christina; Sebat, Jonathan

    2016-01-01

    Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement. PMID:27018473