WorldWideScience

Sample records for genomic dna sequences

  1. Sequencing intractable DNA to close microbial genomes.

    Science.gov (United States)

    Hurt, Richard A; Brown, Steven D; Podar, Mircea; Palumbo, Anthony V; Elias, Dwayne A

    2012-01-01

    Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  2. Sequencing intractable DNA to close microbial genomes.

    Directory of Open Access Journals (Sweden)

    Richard A Hurt

    Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  3. Inconsistencies in Neanderthal genomic DNA sequences.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Wall

    2007-10-01

    Full Text Available Two recently published papers describe nuclear DNA sequences that were obtained from the same Neanderthal fossil. Our reanalyses of the data from these studies show that they are not consistent with each other and point to serious problems with the data quality in one of the studies, possibly due to modern human DNA contaminants and/or a high rate of sequencing errors.

  4. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    coding regions are the DNA uptake sequences (DUS) required for natural genetic transformation. More importantly, we found a significantly higher density of DUS within genes involved in DNA repair, recombination, restriction-modification and replication than in any other annotated gene group......Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within...

  5. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  6. 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...

  7. An automated annotation tool for genomic DNA sequences using GeneScan and BLAST

    Indian Academy of Sciences (India)

    Andrew M. Lynn; Chakresh Kumar Jain; K. Kosalai; Pranjan Barman; Nupur Thakur; Harish Batra; Alok Bhattacharya

    2001-04-01

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

  8. Sequencing of chloroplast genome using whole cellular DNA and Solexa sequencing technology

    Directory of Open Access Journals (Sweden)

    Jian eWu

    2012-11-01

    Full Text Available Sequencing of the chloroplast genome using traditional sequencing methods has been difficult because of its size (>120 kb and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the chloroplast genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassica rapa accessions with one lane per accession. In total, 246 Mb, 362Mb, 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16 and FT, respectively. Microreads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7–99.8% or 95.5–99.7% of the B. rapa chloroplast genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of chloroplast genome.

  9. 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  .

  10. Complete genome sequence of mitochondrial DNA (mtDNA) of Chlorella sorokiniana.

    Science.gov (United States)

    Orsini, Massimiliano; Costelli, Cristina; Malavasi, Veronica; Cusano, Roberto; Concas, Alessandro; Angius, Andrea; Cao, Giacomo

    2016-01-01

    The complete sequence of mitochondrial genome of the Chlorella sorokiniana strain (SAG 111-8 k) is presented in this work. Within the Chlorella genus, it represents the second species with a complete sequenced and annotated mitochondrial genome (GenBank accession no. KM241869). The genome consists of circular chromosomes of 52,528 bp and encodes a total of 31 protein coding genes, 3 rRNAs and 26 tRNAs. The overall AT contents of the C. sorokiniana mtDNA is 70.89%, while the coding sequence is of 97.4%.

  11. Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

    Science.gov (United States)

    Prakash, Ashwin; Bechtel, Jason; Fedorov, Alexei

    2011-01-01

    Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009). Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition. PMID:21610667

  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. Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing.

    Science.gov (United States)

    Sim, James Heng Chiak; Anikst, Victoria; Lohith, Akshar; Pourmand, Nader; Banaei, Niaz

    2015-07-01

    Successful sequencing of the Clostridium difficile genome requires high-quality genomic DNA (gDNA) as the starting material. gDNA extraction using conventional methods is laborious. We describe here an optimized method for the simple extraction of C. difficile gDNA using the QIAamp DNA minikit, which yielded high-quality sequence reads on the Illumina MiSeq platform. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. An improved chloroplast DNA extraction procedure for whole plastid genome sequencing.

    Science.gov (United States)

    Shi, Chao; Hu, Na; Huang, Hui; Gao, Ju; Zhao, You-Jie; Gao, Li-Zhi

    2012-01-01

    Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs. We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40-50% cpDNA purity is achieved with our method. Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

  15. Complete genome sequence of chloroplast DNA (cpDNA) of Chlorella sorokiniana.

    Science.gov (United States)

    Orsini, Massimiliano; Cusano, Roberto; Costelli, Cristina; Malavasi, Veronica; Concas, Alessandro; Angius, Andrea; Cao, Giacomo

    2016-01-01

    The complete chloroplast genome sequence of Chlorella sorokiniana strain (SAG 111-8 k) is presented in this study. The genome consists of circular chromosomes of 109,811 bp, which encode a total of 109 genes, including 74 proteins, 3 rRNAs and 31 tRNAs. Moreover, introns are not detected and all genes are present in single copy. The overall AT contents of the C. sorokiniana cpDNA is 65.9%, the coding sequence is 59.1% and a large inverted repeat (IR) is not observed.

  16. Multi-scale coding of genomic information: From DNA sequence to genome structure and function

    Energy Technology Data Exchange (ETDEWEB)

    Arneodo, Alain, E-mail: alain.arneodo@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Vaillant, Cedric, E-mail: cedric.vaillant@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Audit, Benjamin, E-mail: benjamin.audit@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Argoul, Francoise, E-mail: francoise.argoul@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); D' Aubenton-Carafa, Yves, E-mail: daubenton@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France); Thermes, Claude, E-mail: claude.thermes@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France)

    2011-02-15

    Understanding how chromatin is spatially and dynamically organized in the nucleus of eukaryotic cells and how this affects genome functions is one of the main challenges of cell biology. Since the different orders of packaging in the hierarchical organization of DNA condition the accessibility of DNA sequence elements to trans-acting factors that control the transcription and replication processes, there is actually a wealth of structural and dynamical information to learn in the primary DNA sequence. In this review, we show that when using concepts, methodologies, numerical and experimental techniques coming from statistical mechanics and nonlinear physics combined with wavelet-based multi-scale signal processing, we are able to decipher the multi-scale sequence encoding of chromatin condensation-decondensation mechanisms that play a fundamental role in regulating many molecular processes involved in nuclear functions.

  17. DNA sequencing leads to genomics progress in China

    Institute of Scientific and Technical Information of China (English)

    WU JiaYan; XIAO JingFa; ZHANG RuoSi; YU Jun

    2011-01-01

    1 Science in the large-scale sequencing era Ten years ago,the first draft sequence assembly of the human genome was completed [1],bringing biomedical research one-step closer toward the goal of revolutionizing diagnosis,prevention,and treatment of human diseases.Recently,journalists from the journal Nature surveyed more than 1000 life scientists regarding this laudable aim [2],obtaining substantially negative responses [3].However,almost all of those surveyed had been influenced,in one way or another,by the availability of the human genome sequence,and they also agreed with the notion that the "sequence is the start." The complexity of genome biology and almost every aspect of human biology is far greater than previously thought [4].

  18. How evolution of genomes is reflected in exact DNA sequence match statistics.

    Science.gov (United States)

    Massip, Florian; Sheinman, Michael; Schbath, Sophie; Arndt, Peter F

    2015-02-01

    Genome evolution is shaped by a multitude of mutational processes, including point mutations, insertions, and deletions of DNA sequences, as well as segmental duplications. These mutational processes can leave distinctive qualitative marks in the statistical features of genomic DNA sequences. One such feature is the match length distribution (MLD) of exactly matching sequence segments within an individual genome or between the genomes of related species. These have been observed to exhibit characteristic power law decays in many species. Here, we show that simple dynamical models consisting solely of duplication and mutation processes can already explain the characteristic features of MLDs observed in genomic sequences. Surprisingly, we find that these features are largely insensitive to details of the underlying mutational processes and do not necessarily rely on the action of natural selection. Our results demonstrate how analyzing statistical features of DNA sequences can help us reveal and quantify the different mutational processes that underlie genome evolution.

  19. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.

    Science.gov (United States)

    Mayjonade, Baptiste; Gouzy, Jérôme; Donnadieu, Cécile; Pouilly, Nicolas; Marande, William; Callot, Caroline; Langlade, Nicolas; Muños, Stéphane

    2016-10-01

    De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.

  20. Targeted enrichment of genomic DNA regions for next generation sequencing

    NARCIS (Netherlands)

    Mertens, F.; El-Sharawy, A.; Sauer, S.; Van Helvoort, J.; Van der Zaag, P.J.; Franke, A.; Nilsson, M.; Lehrach. H.; Brookes, A.

    2011-01-01

    In this review we discuss the latest targeted enrichment methods, and aspects of their utilization along with second generation sequencing for complex genome analysis. In doing so we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a pow

  1. An improved chloroplast DNA extraction procedure for whole plastid genome sequencing.

    Directory of Open Access Journals (Sweden)

    Chao Shi

    Full Text Available BACKGROUND: Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs. METHODOLOGY/PRINCIPAL FINDINGS: We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40-50% cpDNA purity is achieved with our method. CONCLUSION: Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

  2. High-throughput DNA sequencing: a genomic data manufacturing process.

    Science.gov (United States)

    Huang, G M

    1999-01-01

    The progress trends in automated DNA sequencing operation are reviewed. Technological development in sequencing instruments, enzymatic chemistry and robotic stations has resulted in ever-increasing capacity of sequence data production. This progress leads to a higher demand on laboratory information management and data quality assessment. High-throughput laboratories face the challenge of organizational management, as well as technology management. Engineering principles of process control should be adopted in this biological data manufacturing procedure. While various systems attempt to provide solutions to automate different parts of, or even the entire process, new technical advances will continue to change the paradigm and provide new challenges.

  3. Origin of noncoding DNA sequences: molecular fossils of genome evolution.

    OpenAIRE

    Naora, H.; MIYAHARA, K.; Curnow, R. N.

    1987-01-01

    The total amount of noncoding sequences on chromosomes of contemporary organisms varies significantly from species to species. We propose a hypothesis for the origin of these noncoding sequences that assumes that (i) an approximately equal to 0.55-kilobase (kb)-long reading frame composed the primordial gene and (ii) a 20-kb-long single-stranded polynucleotide is the longest molecule (as a genome) that was polymerized at random and without a specific template in the primordial soup/cell. The ...

  4. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it po

  5. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome.

    Science.gov (United States)

    Ley, Timothy J; Mardis, Elaine R; Ding, Li; Fulton, Bob; McLellan, Michael D; Chen, Ken; Dooling, David; Dunford-Shore, Brian H; McGrath, Sean; Hickenbotham, Matthew; Cook, Lisa; Abbott, Rachel; Larson, David E; Koboldt, Dan C; Pohl, Craig; Smith, Scott; Hawkins, Amy; Abbott, Scott; Locke, Devin; Hillier, Ladeana W; Miner, Tracie; Fulton, Lucinda; Magrini, Vincent; Wylie, Todd; Glasscock, Jarret; Conyers, Joshua; Sander, Nathan; Shi, Xiaoqi; Osborne, John R; Minx, Patrick; Gordon, David; Chinwalla, Asif; Zhao, Yu; Ries, Rhonda E; Payton, Jacqueline E; Westervelt, Peter; Tomasson, Michael H; Watson, Mark; Baty, Jack; Ivanovich, Jennifer; Heath, Sharon; Shannon, William D; Nagarajan, Rakesh; Walter, Matthew J; Link, Daniel C; Graubert, Timothy A; DiPersio, John F; Wilson, Richard K

    2008-11-06

    Acute myeloid leukaemia is a highly malignant haematopoietic tumour that affects about 13,000 adults in the United States each year. The treatment of this disease has changed little in the past two decades, because most of the genetic events that initiate the disease remain undiscovered. Whole-genome sequencing is now possible at a reasonable cost and timeframe to use this approach for the unbiased discovery of tumour-specific somatic mutations that alter the protein-coding genes. Here we present the results obtained from sequencing a typical acute myeloid leukaemia genome, and its matched normal counterpart obtained from the same patient's skin. We discovered ten genes with acquired mutations; two were previously described mutations that are thought to contribute to tumour progression, and eight were new mutations present in virtually all tumour cells at presentation and relapse, the function of which is not yet known. Our study establishes whole-genome sequencing as an unbiased method for discovering cancer-initiating mutations in previously unidentified genes that may respond to targeted therapies.

  6. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    Directory of Open Access Journals (Sweden)

    Zdepski Anna

    2011-05-01

    Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

  7. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human

    Science.gov (United States)

    Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

    2017-01-01

    DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation. PMID:28212312

  8. Origin of noncoding DNA sequences: molecular fossils of genome evolution.

    Science.gov (United States)

    Naora, H; Miyahara, K; Curnow, R N

    1987-09-01

    The total amount of noncoding sequences on chromosomes of contemporary organisms varies significantly from species to species. We propose a hypothesis for the origin of these noncoding sequences that assumes that (i) an approximately equal to 0.55-kilobase (kb)-long reading frame composed the primordial gene and (ii) a 20-kb-long single-stranded polynucleotide is the longest molecule (as a genome) that was polymerized at random and without a specific template in the primordial soup/cell. The statistical distribution of stop codons allows examination of the probability of generating reading frames of approximately equal to 0.55 kb in this primordial polynucleotide. This analysis reveals that with three stop codons, a run of at least 0.55-kb equivalent length of nonstop codons would occur in 4.6% of 20-kb-long polynucleotide molecules. We attempt to estimate the total amount of noncoding sequences that would be present on the chromosomes of contemporary species assuming that present-day chromosomes retain the prototype primordial genome structure. Theoretical estimates thus obtained for most eukaryotes do not differ significantly from those reported for these specific organisms, with only a few exceptions. Furthermore, analysis of possible stop-codon distributions suggests that life on earth would not exist, at least in its present form, had two or four stop codons been selected early in evolution.

  9. Origin of noncoding DNA sequences: molecular fossils of genome evolution

    Energy Technology Data Exchange (ETDEWEB)

    Naora, H.; Miyahara, K.; Curnow, R.N.

    1987-09-01

    The total amount of noncoding sequences on chromosomes of contemporary organisms varies significantly from species to species. The authors propose a hypothesis for the origin of these noncoding sequences that assumes that (i) an approx. 0.55-kilobase (kb)-long reading frame composed the primordial gene and (ii) a 20-kb-long single-stranded polynucleotide is the longest molecule (as a genome) that was polymerized at random and without a specific template in the primordial soup/cell. The statistical distribution of stop codons allows examination of the probability of generating reading frames of approx. 0.55 kb in this primordial polynucleotide. This analysis reveals that with three stop codons, a run of at least 0.55-kb equivalent length of nonstop codons would occur in 4.6% of 20-kb-long polynucleotide molecules. They attempt to estimate the total amount of noncoding sequences that would be present on the chromosomes of contemporary species assuming that present-day chromosomes retain the prototype primordial genome structure. Theoretical estimates thus obtained for most eukaryotes do not differ significantly from those reported for these specific organisms, with only a few exceptions. Furthermore, analysis of possible stop-codon distributions suggests that life on earth would not exist, at least in its present form, had two or four stop codons been selected early in evolution.

  10. Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    Directory of Open Access Journals (Sweden)

    Momchilo Vuyisich

    2014-01-01

    Full Text Available Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg. There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderia spp., which have the highest GC content and are the longest, we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic DNA is used.

  11. Genomic and polyploid evolution in genus Avena as revealed by RFLPs of repeated DNA sequences.

    Science.gov (United States)

    Morikawa, Toshinobu; Nishihara, Miho

    2009-06-01

    Phylogenetic relationships and genome affinities were investigated by utilizing all the biological Avena species consisting of 11 diploid species (15 accessions), 8 tetraploid species (9 accessions) and 4 hexaploid species (5 accessions). Genomic DNA regions of As120a, avenin, and globulin were amplified by PCR. A total of 130 polymorphic fragments were detected out of 156 fragments generated by digesting the PCR-amplified fragments with 11 restriction enzymes. The number of fragments generated by PCR-amplification followed by digestion with restriction enzymes was almost the same as those among the three repeated DNA sequences. A high level of genetic distance was detected between A. damascena (Ad) and A. canariensis (Ac) genomes, which reflected their different morphology and reproductive isolation. The A. longiglumis (Al) and A. prostrata (Ap) genomes were closely related to the As genome group. The AB genome species formed a cluster with the AsAs genome artificial autotetraploid and the As genome diploids indicating near-autotetraploid origin. The A. macrostachya is an outbreeding autotetraploid closely related with the C genome diploid and the AC genome tetraploid species. The differences of genetic distances estimated from the repeated DNA sequence divergence among the Avena species were consistent with genome divergences and it was possible to compare the genetic intra- and inter-ploidy relationships produced by RFLPs. These results suggested that the PCR-mediated analysis of repeated DNA polymorphism can be used as a tool to examine genomic relationships of polyploidy species.

  12. Ribosomal DNA sequence heterogeneity reflects intraspecies phylogenies and predicts genome structure in two contrasting yeast species.

    Science.gov (United States)

    West, Claire; James, Stephen A; Davey, Robert P; Dicks, Jo; Roberts, Ian N

    2014-07-01

    The ribosomal RNA encapsulates a wealth of evolutionary information, including genetic variation that can be used to discriminate between organisms at a wide range of taxonomic levels. For example, the prokaryotic 16S rDNA sequence is very widely used both in phylogenetic studies and as a marker in metagenomic surveys and the internal transcribed spacer region, frequently used in plant phylogenetics, is now recognized as a fungal DNA barcode. However, this widespread use does not escape criticism, principally due to issues such as difficulties in classification of paralogous versus orthologous rDNA units and intragenomic variation, both of which may be significant barriers to accurate phylogenetic inference. We recently analyzed data sets from the Saccharomyces Genome Resequencing Project, characterizing rDNA sequence variation within multiple strains of the baker's yeast Saccharomyces cerevisiae and its nearest wild relative Saccharomyces paradoxus in unprecedented detail. Notably, both species possess single locus rDNA systems. Here, we use these new variation datasets to assess whether a more detailed characterization of the rDNA locus can alleviate the second of these phylogenetic issues, sequence heterogeneity, while controlling for the first. We demonstrate that a strong phylogenetic signal exists within both datasets and illustrate how they can be used, with existing methodology, to estimate intraspecies phylogenies of yeast strains consistent with those derived from whole-genome approaches. We also describe the use of partial Single Nucleotide Polymorphisms, a type of sequence variation found only in repetitive genomic regions, in identifying key evolutionary features such as genome hybridization events and show their consistency with whole-genome Structure analyses. We conclude that our approach can transform rDNA sequence heterogeneity from a problem to a useful source of evolutionary information, enabling the estimation of highly accurate phylogenies of

  13. CLONING AND ANALYSIS OF THE GENOMIC DNA SEQUENCE OF AUGMENTER OF LIVER REGENERATION FROM RAT

    Institute of Scientific and Technical Information of China (English)

    董菁; 成军; 王勤环; 施双双; 王刚; 斯崇文

    2002-01-01

    Objective.To search for genomic DNA sequence of the augmenter of liver regeneration (ALR) of rat.Methods.Polymerase chain reaction (PCR) with specific primers was used to amplify the sequence from the rat genome.Results.A piece of genomic DNA sequence and a piece of pseudogene of rat ALR were identified.The lengths of the gene and pseudogene are 1508 bp and 442 bp,respectively.The ALR gene of rat includes 3 exons and 2 introns.The 442 bp DNA sequence may represent a pseudogene or a ALR related peptide.Predicted amino acid sequence analysis showed that there were 14 different amino acid residues between the gene and pseudogene.ALR related peptide is 84 amino acid residues in length and relates closely to ALR protein.Conclusion.There might be a multigene family of ALR in rat.

  14. High-throughput sequencing of three Lemnoideae (duckweeds chloroplast genomes from total DNA.

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    Wenqin Wang

    Full Text Available BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

  15. Complete Genome Sequence of the WHO International Standard for Hepatitis B Virus DNA

    Science.gov (United States)

    Jenkins, Adrian; Minhas, Rehan

    2017-01-01

    ABSTRACT   The World Health Organization (WHO) international standard (IS) for hepatitis B virus (HBV) DNA for use in nucleic acid amplification assays was characterized by determining the complete genome sequence, which was assigned genotype A. This information will aid the design, development, and evaluation of HBV DNA amplification assays. PMID:28209818

  16. Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

    DEFF Research Database (Denmark)

    Nosek, J.; Novotna, M.; Hlavatovicova, Z.

    2004-01-01

    The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population...

  17. Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

    DEFF Research Database (Denmark)

    Nosek, J.; Novotna, M.; Hlavatovicova, Z.

    2004-01-01

    The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population...

  18. Complete genome sequence of the mitochondrial DNA of the river lamprey, Lethenteron japonicum.

    Science.gov (United States)

    Kawai, Yuri L; Yura, Kei; Shindo, Miyuki; Kusakabe, Rie; Hayashi, Keiko; Hata, Kenichiro; Nakabayashi, Kazuhiko; Okamura, Kohji

    2015-01-01

    Lampreys are eel-like jawless fishes evolutionarily positioned between invertebrates and vertebrates, and have been used as model organisms to explore vertebrate evolution. In this study we determined the complete genome sequence of the mitochondrial DNA of the Japanese river lamprey, Lethenteron japonicum, using next-generation sequencers. The sequence was 16,272 bp in length. The gene content and order were identical to those of the sea lamprey, Petromyzon marinus, which has been the reference among lamprey species. However, the sequence similarity was less than 90%, suggesting the need for the whole-genome sequencing of L. japonicum.

  19. Genomic Signal Processing Methods for Computation of Alignment-Free Distances from DNA Sequences

    Science.gov (United States)

    Borrayo, Ernesto; Mendizabal-Ruiz, E. Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P.; Morales, J. Alejandro

    2014-01-01

    Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments. PMID:25393409

  20. Genomic signal processing methods for computation of alignment-free distances from DNA sequences.

    Science.gov (United States)

    Borrayo, Ernesto; Mendizabal-Ruiz, E Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P; Morales, J Alejandro

    2014-01-01

    Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments.

  1. Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

    Directory of Open Access Journals (Sweden)

    Mohammed Bakkali

    Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

  2. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    Science.gov (United States)

    Afek, Ariel; Cohen, Hila; Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B

    2015-08-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  3. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    Directory of Open Access Journals (Sweden)

    Ariel Afek

    2015-08-01

    Full Text Available Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large

  4. DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

    Science.gov (United States)

    Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

    2016-01-01

    The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

  5. Genome Sequencing

    DEFF Research Database (Denmark)

    Sato, Shusei; Andersen, Stig Uggerhøj

    2014-01-01

    The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based on transcr......The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based...

  6. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    Directory of Open Access Journals (Sweden)

    Tran Duc

    2010-05-01

    Full Text Available Abstract Background Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the

  7. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

    2010-05-07

    Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

  8. Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data.

    Directory of Open Access Journals (Sweden)

    Can Alkan

    2007-09-01

    Full Text Available The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%-5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.

  9. Genomic DNA enrichment using sequence capture microarrays: a novel approach to discover sequence nucleotide polymorphisms (SNP in Brassica napus L.

    Directory of Open Access Journals (Sweden)

    Wayne E Clarke

    Full Text Available Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38. The main goal of this project was to combine sequence capture with next generation sequencing (NGS to discover single nucleotide polymorphisms (SNPs in specific areas of the B. napus genome historically associated (via quantitative trait loci -QTL- analysis to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively. Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species.

  10. High-throughput DNA Stretching in Continuous Elongational Flow for Genome Sequence Scanning

    Science.gov (United States)

    Meltzer, Robert; Griffis, Joshua; Safranovitch, Mikhail; Malkin, Gene; Cameron, Douglas

    2014-03-01

    Genome Sequence Scanning (GSS) identifies and compares bacterial genomes by stretching long (60 - 300 kb) genomic DNA restriction fragments and scanning for site-selective fluorescent probes. Practical application of GSS requires: 1) high throughput data acquisition, 2) efficient DNA stretching, 3) reproducible DNA elasticity in the presence of intercalating fluorescent dyes. GSS utilizes a pseudo-two-dimensional micron-scale funnel with convergent sheathing flows to stretch one molecule at a time in continuous elongational flow and center the DNA stream over diffraction-limited confocal laser excitation spots. Funnel geometry has been optimized to maximize throughput of DNA within the desired length range (>10 million nucleobases per second). A constant-strain detection channel maximizes stretching efficiency by applying a constant parabolic tension profile to each molecule, minimizing relaxation and flow-induced tumbling. The effect of intercalator on DNA elasticity is experimentally controlled by reacting one molecule of DNA at a time in convergent sheathing flows of the dye. Derivations of accelerating flow and non-linear tension distribution permit alignment of detected fluorescence traces to theoretical templates derived from whole-genome sequence data.

  11. Methylated DNA is over-represented in whole-genome bisulfite sequencing data

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    Lexiang eJi

    2014-10-01

    Full Text Available The development of whole-genome bisulfite sequencing (WGBS has led to a number of exciting discoveries about the role of DNA methylation leading to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently advanced to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of reads from methylated DNA in WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. It is especially critical for studies that seek to accurately quantify DNA methylation levels in populations that may segregate for allelic DNA methylation states.

  12. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H. influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions....

  13. Genome evolution and meiotic maps by massively parallel DNA sequencing: spotted gar, an outgroup for the teleost genome duplication.

    Science.gov (United States)

    Amores, Angel; Catchen, Julian; Ferrara, Allyse; Fontenot, Quenton; Postlethwait, John H

    2011-08-01

    Genomic resources for hundreds of species of evolutionary, agricultural, economic, and medical importance are unavailable due to the expense of well-assembled genome sequences and difficulties with multigenerational studies. Teleost fish provide many models for human disease but possess anciently duplicated genomes that sometimes obfuscate connectivity. Genomic information representing a fish lineage that diverged before the teleost genome duplication (TGD) would provide an outgroup for exploring the mechanisms of evolution after whole-genome duplication. We exploited massively parallel DNA sequencing to develop meiotic maps with thrift and speed by genotyping F(1) offspring of a single female and a single male spotted gar (Lepisosteus oculatus) collected directly from nature utilizing only polymorphisms existing in these two wild individuals. Using Stacks, software that automates the calling of genotypes from polymorphisms assayed by Illumina sequencing, we constructed a map containing 8406 markers. RNA-seq on two map-cross larvae provided a reference transcriptome that identified nearly 1000 mapped protein-coding markers and allowed genome-wide analysis of conserved synteny. Results showed that the gar lineage diverged from teleosts before the TGD and its genome is organized more similarly to that of humans than teleosts. Thus, spotted gar provides a critical link between medical models in teleost fish, to which gar is biologically similar, and humans, to which gar is genomically similar. Application of our F(1) dense mapping strategy to species with no prior genome information promises to facilitate comparative genomics and provide a scaffold for ordering the numerous contigs arising from next generation genome sequencing.

  14. Detection of genomic variation by selection of a 9 mb DNA region and high throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Sergey I Nikolaev

    Full Text Available Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb and 7 (1.1 Mb from an individual from the International HapMap Project (NA12872. We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage > or = 4-fold, and 97.9% concordant in regions with coverage > or = 15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

  15. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  16. Genome-scale DNA sequence recognition by hybridization to short oligomers.

    Science.gov (United States)

    Milosavljević, A; Savković, S; Crkvenjakov, R; Salbego, D; Serrato, H; Kreuzer, H; Gemmell, A; Batus, S; Grujić, D; Carnahan, S; Tepavcević, J

    1996-01-01

    Recently developed hybridization technology (Drmanac et al. 1994) enables economical large-scale detection of short oligomers within DNA fragments. The newly developed recognition method (Milosavljević 1995b) enables comparison of lists of oligomers detected within DNA fragments against known DNA sequences. We here describe an experiment involving a set of 4,513 distinct genomic E.coli clones of average length 2kb, each hybridized with 636 randomly selected short oligomer probes. High hybridization signal with a particular probe was used as an indication of the presence of a complementary oligomer in the particular clone. For each clone, a list of oligomers with highest hybridization signals was compiled. The database consisting of 4,513 oligomer lists was then searched using known E.coli sequences as queries in an attempt to identify the clones that match the query sequence. Out of a total of 11 clones that were recognized at highest significance level by our method, 8 were single-pass sequenced from both ends. The single-pass sequenced ends were then compared against the query sequences. The sequence comparisons confirmed 7 out of the total of 8 examined recognitions. This experiment represents the first successful example of genome-scale sequence recognition based on hybridization data.

  17. Genomic shotgun array: a procedure linking large-scale DNA sequencing with regional transcript mapping.

    Science.gov (United States)

    Li, Ling-Hui; Li, Jian-Chiuan; Lin, Yung-Feng; Lin, Chung-Yen; Chen, Chung-Yung; Tsai, Shih-Feng

    2004-02-11

    To facilitate transcript mapping and to investigate alterations in genomic structure and gene expression in a defined genomic target, we developed a novel microarray-based method to detect transcriptional activity of the human chromosome 4q22-24 region. Loss of heterozygosity of human 4q22-24 is frequently observed in hepatocellular carcinoma (HCC). One hundred and eighteen well-characterized genes have been identified from this region. We took previously sequenced shotgun subclones as templates to amplify overlapping sequences for the genomic segment and constructed a chromosome-region-specific microarray. Using genomic DNA fragments as probes, we detected transcriptional activity from within this region among five different tissues. The hybridization results indicate that there are new transcripts that have not yet been identified by other methods. The existence of new transcripts encoded by genes in this region was confirmed by PCR cloning or cDNA library screening. The procedure reported here allows coupling of shotgun sequencing with transcript mapping and, potentially, detailed analysis of gene expression and chromosomal copy of the genomic sequence for the putative HCC tumor suppressor gene(s) in the 4q candidate region.

  18. The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells.

    Science.gov (United States)

    Murray, Vincent; Chen, Jon K; Tanaka, Mark M

    2016-07-01

    The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5'-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5'-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5'-GT*A and 5'- TGT* trinucleotide sequences, and 5'-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5'-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine-pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the -3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.

  19. DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria

    Directory of Open Access Journals (Sweden)

    Sabin Robert

    2002-12-01

    Full Text Available Abstract Background Complete sequencing and annotation of the 96.2 kb Bacillus anthracis plasmid, pXO2, predicted 85 open reading frames (ORFs. Bacillus cereus and Bacillus thuringiensis isolates that ranged in genomic similarity to B. anthracis, as determined by amplified fragment length polymorphism (AFLP analysis, were examined by PCR for the presence of sequences similar to 47 pXO2 ORFs. Results The two most distantly related isolates examined, B. thuringiensis 33679 and B. thuringiensis AWO6, produced the greatest number of ORF sequences similar to pXO2; 10 detected in 33679 and 16 in AWO6. No more than two of the pXO2 ORFs were detected in any one of the remaining isolates. Dot-blot DNA hybridizations between pXO2 ORF fragments and total genomic DNA from AWO6 were consistent with the PCR assay results for this isolate and also revealed nine additional ORFs shared between these two bacteria. Sequences similar to the B. anthracis cap genes or their regulator, acpA, were not detected among any of the examined isolates. Conclusions The presence of pXO2 sequences in the other Bacillus isolates did not correlate with genomic relatedness established by AFLP analysis. The presence of pXO2 ORF sequences in other Bacillus species suggests the possibility that certain pXO2 plasmid gene functions may also be present in other closely related bacteria.

  20. Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

    Directory of Open Access Journals (Sweden)

    Chang Su

    2014-12-01

    Full Text Available DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees.

  1. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.

    Science.gov (United States)

    Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

    2013-09-24

    Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

  2. Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

    Directory of Open Access Journals (Sweden)

    Minsoung Rhee

    Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

  3. Roche genome sequencer FLX based high-throughput sequencing of ancient DNA

    DEFF Research Database (Denmark)

    Alquezar-Planas, David E; Fordyce, Sarah Louise

    2012-01-01

    Since the development of so-called "next generation" high-throughput sequencing in 2005, this technology has been applied to a variety of fields. Such applications include disease studies, evolutionary investigations, and ancient DNA. Each application requires a specialized protocol to ensure tha...

  4. Sequence to Medical Phenotypes: A Framework for Interpretation of Human Whole Genome DNA Sequence Data.

    Directory of Open Access Journals (Sweden)

    Frederick E Dewey

    2015-10-01

    Full Text Available High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework.

  5. DNA sequence explains seemingly disordered methylation levels in partially methylated domains of Mammalian genomes.

    Directory of Open Access Journals (Sweden)

    Dimos Gaidatzis

    2014-02-01

    Full Text Available For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs, recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40% of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS. Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2 of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

  6. DNA immunoprecipitation semiconductor sequencing (DIP-SC-seq) as a rapid method to generate genome wide epigenetic signatures.

    Science.gov (United States)

    Thomson, John P; Fawkes, Angie; Ottaviano, Raffaele; Hunter, Jennifer M; Shukla, Ruchi; Mjoseng, Heidi K; Clark, Richard; Coutts, Audrey; Murphy, Lee; Meehan, Richard R

    2015-05-14

    Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; "DIP-SC-seq") on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets.

  7. DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly

    NARCIS (Netherlands)

    Scheinin, I.; Sie, D.; Bengtsson, H.; Wiel, M.A. van de; Olshen, A.B.; Thuijl, H.F. van; Essen, H.F. van; Eijk, P.P.; Rustenburg, F.; Meijer, G.A.; Reijneveld, J.C.; Wesseling, P.; Pinkel, D.; Albertson, D.G.; Ylstra, B.

    2014-01-01

    Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraff

  8. Genomic organization and dynamics of repetitive DNA sequences in representatives of three Fagaceae genera.

    Science.gov (United States)

    Alves, Sofia; Ribeiro, Teresa; Inácio, Vera; Rocheta, Margarida; Morais-Cecílio, Leonor

    2012-05-01

    Oaks, chestnuts, and beeches are economically important species of the Fagaceae. To understand the relationship between these members of this family, a deep knowledge of their genome composition and organization is needed. In this work, we have isolated and characterized several AFLP fragments obtained from Quercus rotundifolia Lam. through homology searches in available databases. Genomic polymorphisms involving some of these sequences were evaluated in two species of Quercus, one of Castanea, and one of Fagus with specific primers. Comparative FISH analysis with generated sequences was performed in interphase nuclei of the four species, and the co-immunolocalization of 5-methylcytosine was also studied. Some of the sequences isolated proved to be genus-specific, while others were present in all the genera. Retroelements, either gypsy-like of the Tat/Athila clade or copia-like, are well represented, and most are dispersed in euchromatic regions of these species with no DNA methylation associated, pointing to an interspersed arrangement of these retroelements with potential gene-rich regions. A particular gypsy-sequence is dispersed in oaks and chestnut nuclei, but its confinement to chromocenters in beech evidences genome restructuring events during evolution of Fagaceae. Several sequences generated in this study proved to be good tools to comparatively study Fagaceae genome organization.

  9. Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

    Directory of Open Access Journals (Sweden)

    Andaine Seguin-Orlando

    Full Text Available Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

  10. Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

    Science.gov (United States)

    Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel; Stagegaard, Julia; Alberdi, Maria T; Prado, José Luis; Prieto, Alfredo; Willerslev, Eske; Orlando, Ludovic

    2013-01-01

    Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

  11. Seventeen new complete mtDNA sequences reveal extensive mitochondrial genome evolution within the Demospongiae.

    Directory of Open Access Journals (Sweden)

    Xiujuan Wang

    Full Text Available Two major transitions in animal evolution--the origins of multicellularity and bilaterality--correlate with major changes in mitochondrial DNA (mtDNA organization. Demosponges, the largest class in the phylum Porifera, underwent only the first of these transitions and their mitochondrial genomes display a peculiar combination of ancestral and animal-specific features. To get an insight into the evolution of mitochondrial genomes within the Demospongiae, we determined 17 new mtDNA sequences from this group and analyzing them with five previously published sequences. Our analysis revealed that all demosponge mtDNAs are 16- to 25-kbp circular molecules, containing 13-15 protein genes, 2 rRNA genes, and 2-27 tRNA genes. All but four pairs of sampled genomes had unique gene orders, with the number of shared gene boundaries ranging from 1 to 41. Although most demosponge species displayed low rates of mitochondrial sequence evolution, a significant acceleration in evolutionary rates occurred in the G1 group (orders Dendroceratida, Dictyoceratida, and Verticillitida. Large variation in mtDNA organization was also observed within the G0 group (order Homosclerophorida including gene rearrangements, loss of tRNA genes, and the presence of two introns in Plakortis angulospiculatus. While introns are rare in modern-day demosponge mtDNA, we inferred that at least one intron was present in cox1 of the common ancestor of all demosponges. Our study uncovered an extensive mitochondrial genomic diversity within the Demospongiae. Although all sampled mitochondrial genomes retained some ancestral features, including a minimally modified genetic code, conserved structures of tRNA genes, and presence of multiple non-coding regions, they vary considerably in their size, gene content, gene order, and the rates of sequence evolution. Some of the changes in demosponge mtDNA, such as the loss of tRNA genes and the appearance of hairpin-containing repetitive elements

  12. Seventeen New Complete mtDNA Sequences Reveal Extensive Mitochondrial Genome Evolution within the Demospongiae

    Science.gov (United States)

    Wang, Xiujuan; Lavrov, Dennis V.

    2008-01-01

    Two major transitions in animal evolution–the origins of multicellularity and bilaterality–correlate with major changes in mitochondrial DNA (mtDNA) organization. Demosponges, the largest class in the phylum Porifera, underwent only the first of these transitions and their mitochondrial genomes display a peculiar combination of ancestral and animal-specific features. To get an insight into the evolution of mitochondrial genomes within the Demospongiae, we determined 17 new mtDNA sequences from this group and analyzing them with five previously published sequences. Our analysis revealed that all demosponge mtDNAs are 16- to 25-kbp circular molecules, containing 13–15 protein genes, 2 rRNA genes, and 2–27 tRNA genes. All but four pairs of sampled genomes had unique gene orders, with the number of shared gene boundaries ranging from 1 to 41. Although most demosponge species displayed low rates of mitochondrial sequence evolution, a significant acceleration in evolutionary rates occurred in the G1 group (orders Dendroceratida, Dictyoceratida, and Verticillitida). Large variation in mtDNA organization was also observed within the G0 group (order Homosclerophorida) including gene rearrangements, loss of tRNA genes, and the presence of two introns in Plakortis angulospiculatus. While introns are rare in modern-day demosponge mtDNA, we inferred that at least one intron was present in cox1 of the common ancestor of all demosponges. Our study uncovered an extensive mitochondrial genomic diversity within the Demospongiae. Although all sampled mitochondrial genomes retained some ancestral features, including a minimally modified genetic code, conserved structures of tRNA genes, and presence of multiple non-coding regions, they vary considerably in their size, gene content, gene order, and the rates of sequence evolution. Some of the changes in demosponge mtDNA, such as the loss of tRNA genes and the appearance of hairpin-containing repetitive elements, occurred in

  13. Complete DNA sequences of the mitochondrial genomes of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis: insight into the evolution of linear DNA genomes from mitochondrial telomere mutants.

    Science.gov (United States)

    Kosa, Peter; Valach, Matus; Tomaska, Lubomir; Wolfe, Kenneth H; Nosek, Jozef

    2006-01-01

    We determined complete mitochondrial DNA sequences of the two yeast species, Candida orthopsilosis and Candida metapsilosis, and compared them with the linear mitochondrial genome of their close relative, C.parapsilosis. Mitochondria of all the three species harbor compact genomes encoding the same set of genes arranged in the identical order. Differences in the length of these genomes result mainly from the presence/absence of introns. Multiple alterations were identified also in the sequences of the ribosomal and transfer RNAs, and proteins. However, the most striking feature of C.orthopsilosis and C.metapsilosis is the existence of strains differing in the molecular form of the mitochondrial genome (circular-mapping versus linear). Their analysis opens a unique window for understanding the role of mitochondrial telomeres in the stability and evolution of molecular architecture of the genome. Our results indicate that the circular-mapping mitochondrial genome derived from the linear form by intramolecular end-to-end fusions. Moreover, we suggest that the linear mitochondrial genome evolved from a circular-mapping form present in a common ancestor of the three species and, at the same time, the emergence of mitochondrial telomeres enabled the formation of linear monomeric DNA forms. In addition, comparison of isogenic C.metapsilosis strains differing in the form of the organellar genome suggests a possibility that, under some circumstances, the linearity and/or the presence of telomeres provide a competitive advantage over a circular-mapping mitochondrial genome.

  14. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave; Lescai, Francesco; Grove, Jakob

    2016-01-01

    Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can......_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate...... be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject...

  15. Complete sequence of the mitochondrial genome of Tetrahymena pyriformis and comparison with Paramecium aurelia mitochondrial DNA.

    Science.gov (United States)

    Burger, G; Zhu, Y; Littlejohn, T G; Greenwood, S J; Schnare, M N; Lang, B F; Gray, M W

    2000-03-24

    We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events

  16. Locus Reference Genomic sequences: An improved basis for describing human DNA variants

    KAUST Repository

    Dalgleish, Raymond

    2010-04-15

    As our knowledge of the complexity of gene architecture grows, and we increase our understanding of the subtleties of gene expression, the process of accurately describing disease-causing gene variants has become increasingly problematic. In part, this is due to current reference DNA sequence formats that do not fully meet present needs. Here we present the Locus Reference Genomic (LRG) sequence format, which has been designed for the specifi c purpose of gene variant reporting. The format builds on the successful National Center for Biotechnology Information (NCBI) RefSeqGene project and provides a single-fi le record containing a uniquely stable reference DNA sequence along with all relevant transcript and protein sequences essential to the description of gene variants. In principle, LRGs can be created for any organism, not just human. In addition, we recognize the need to respect legacy numbering systems for exons and amino acids and the LRG format takes account of these. We hope that widespread adoption of LRGs - which will be created and maintained by the NCBI and the European Bioinformatics Institute (EBI) - along with consistent use of the Human Genome Variation Society (HGVS)- approved variant nomenclature will reduce errors in the reporting of variants in the literature and improve communication about variants aff ecting human health. Further information can be found on the LRG web site (http://www.lrg-sequence.org). 2010 Dalgleish et al.; licensee BioMed Central Ltd.

  17. Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing.

    Science.gov (United States)

    Bjorn-Mortensen, K; Zallet, J; Lillebaek, T; Andersen, A B; Niemann, S; Rasmussen, E M; Kohl, T A

    2015-08-01

    Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

    DEFF Research Database (Denmark)

    Bjorn-Mortensen, K; Zallet, J; Lillebaek, T

    2015-01-01

    Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable...

  19. Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

    Science.gov (United States)

    Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

    2016-02-25

    Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

  20. Tumorigenic poxviruses: genomic organization and DNA sequence of the telomeric region of the Shope fibroma virus genome.

    Science.gov (United States)

    Upton, C; DeLange, A M; McFadden, G

    1987-09-01

    Shope fibroma virus (SFV), a tumorigenic poxvirus, has a 160-kb linear double-stranded DNA genome and possesses terminal inverted repeats (TIRs) of 12.4 kb. The DNA sequence of the terminal 5.5 kb of the viral genome is presented and together with previously published sequences completes the entire sequence of the SFV TIR. The terminal 400-bp region contains no major open reading frames (ORFs) but does possess five related imperfect palindromes. The remaining 5.1 kb of the sequence contains seven tightly clustered and tandemly oriented ORFs, four larger than 100 amino acids in length (T1, T2, T4, and T5) and three smaller ORFs (T3A, T3B, and T3C). All are transcribed toward the viral hairpin and almost all possess the consensus sequence TTTTTNT near their 3' ends which has been implicated for the transcription termination of vaccinia virus early genes. Searches of the published DNA database revealed no sequences with significant homology with this region of the SFV genome but when the protein database was searched with the translation products of ORFs T1-T5 it was found that the N-terminus of the putative T4 polypeptide is closely related to the signal sequence of the hemagglutinin precursor from influenza A virus, suggesting that the T4 polypeptide may be secreted from SFV-infected cells. Examination of other SFV ORFs shows that T1 and T2 also possess signal-like hydrophobic amino acid stretches close to their N-termini. The protein database search also revealed that the putative T2 protein has significant homology to the insulin family of polypeptides. In terms of sequence repetitions, seven tandemly repeated copies of the hexanucleotide ATTGTT and three flanking regions of dyad symmetry were detected, all in ORF T3C. A search for palindromic sequences also revealed two clusters, one in ORF T3A/B and a second in ORF T2. ORF T2 harbors five short sequence domains, each of which consists of a 6-bp short palindrome and a 10- to 18-bp larger palindrome. The

  1. Mycobacterial DNA extraction for whole-genome sequencing from early positive liquid (MGIT) cultures.

    Science.gov (United States)

    Votintseva, Antonina A; Pankhurst, Louise J; Anson, Luke W; Morgan, Marcus R; Gascoyne-Binzi, Deborah; Walker, Timothy M; Quan, T Phuong; Wyllie, David H; Del Ojo Elias, Carlos; Wilcox, Mark; Walker, A Sarah; Peto, Tim E A; Crook, Derrick W

    2015-04-01

    We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with 90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools. Copyright © 2015, Votintseva et al.

  2. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA.

    Directory of Open Access Journals (Sweden)

    Jesper Buchhave Poulsen

    Full Text Available Stored neonatal dried blood spot (DBS samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA. Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2 and raw DNA extract of the WB reference sample (WB_ref. Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity-the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference-whole-blood DNA-based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.

  3. A survey of genomic traces reveals a common sequencing error, RNA editing, and DNA editing.

    Directory of Open Access Journals (Sweden)

    Alexander Wait Zaranek

    2010-05-01

    Full Text Available While it is widely held that an organism's genomic information should remain constant, several protein families are known to modify it. Members of the AID/APOBEC protein family can deaminate DNA. Similarly, members of the ADAR family can deaminate RNA. Characterizing the scope of these events is challenging. Here we use large genomic data sets, such as the two billion sequences in the NCBI Trace Archive, to look for clusters of mismatches of the same type, which are a hallmark of editing events caused by APOBEC3 and ADAR. We align 603,249,815 traces from the NCBI trace archive to their reference genomes. In clusters of mismatches of increasing size, at least one systematic sequencing error dominates the results (G-to-A. It is still present in mismatches with 99% accuracy and only vanishes in mismatches at 99.99% accuracy or higher. The error appears to have entered into about 1% of the HapMap, possibly affecting other users that rely on this resource. Further investigation, using stringent quality thresholds, uncovers thousands of mismatch clusters with no apparent defects in their chromatograms. These traces provide the first reported candidates of endogenous DNA editing in human, further elucidating RNA editing in human and mouse and also revealing, for the first time, extensive RNA editing in Xenopus tropicalis. We show that the NCBI Trace Archive provides a valuable resource for the investigation of the phenomena of DNA and RNA editing, as well as setting the stage for a comprehensive mapping of editing events in large-scale genomic datasets.

  4. Dragon polya spotter: Predictor of poly(A) motifs within human genomic DNA sequences

    KAUST Repository

    Kalkatawi, Manal Matoq Saeed

    2011-11-15

    Motivation: Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying poly(A) motifs in the primary genomic DNA sequence that correspond to poly(A) signals in mRNA is a far more challenging problem. Recognition of poly(A) signals is important for better gene annotation and understanding of the gene regulation mechanisms. In this work, we present one such poly(A) motif prediction method based on properties of human genomic DNA sequence surrounding a poly(A) motif. These properties include thermodynamic, physico-chemical and statistical characteristics. For predictions, we developed Artificial Neural Network and Random Forest models. These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity and specificity and furthermore provide a consistent level of accuracy for 12 poly(A) motif variants. The Author(s) 2011. Published by Oxford University Press. All rights reserved.

  5. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries.

    Science.gov (United States)

    Carpenter, Meredith L; Buenrostro, Jason D; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M Thomas P; Willerslev, Eske; Greenleaf, William J; Bustamante, Carlos D

    2013-11-07

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.

  6. Analysis of the Campylobacter jejuni genome by SMRT DNA sequencing identifies restriction-modification motifs.

    Directory of Open Access Journals (Sweden)

    Jason L O'Loughlin

    Full Text Available Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni.

  7. An optimized chloroplast DNA extraction protocol for grasses (Poaceae proves suitable for whole plastid genome sequencing and SNP detection.

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    Kerstin Diekmann

    Full Text Available BACKGROUND: Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge. CONCLUSIONS/SIGNIFICANCE: The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus x giganteus, Panicoideae. The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.

  8. Assessing Genetic Diversity among Brettanomyces Yeasts by DNA Fingerprinting and Whole-Genome Sequencing

    Science.gov (United States)

    Crauwels, Sam; Zhu, Bo; Steensels, Jan; Busschaert, Pieter; De Samblanx, Gorik; Marchal, Kathleen; Willems, Kris A.

    2014-01-01

    Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis. PMID:24814796

  9. Assessing genetic diversity among Brettanomyces yeasts by DNA fingerprinting and whole-genome sequencing.

    Science.gov (United States)

    Crauwels, Sam; Zhu, Bo; Steensels, Jan; Busschaert, Pieter; De Samblanx, Gorik; Marchal, Kathleen; Willems, Kris A; Verstrepen, Kevin J; Lievens, Bart

    2014-07-01

    Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. Complete DNA sequence of Kuraishia capsulata illustrates novel genomic features among budding yeasts (Saccharomycotina)

    NARCIS (Netherlands)

    Morales, L.; Noel, B.; Porcel, B.; Marcet-Houben, M.; Hullo, M.F.; Sacerdot, C.; Tekaia, F.; Leh-Louis, V.; Despons, L.; Khanna, V.; Aury, J.M.; Barbe, V.; Couloux, A.; Labadie, K.; Pelletier, E.; Souciet, J.L.; Boekhout, T.; Gabaldon, T.; Wincker, P.; Dujon, B.

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics, but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid type strain

  11. Complete DNA sequence of Kuraishia capsulata illustrates novel genomic features among budding yeasts (Saccharomycotina)

    NARCIS (Netherlands)

    Morales, L.; Noel, B.; Porcel, B.; Marcet-Houben, M.; Hullo, M.F.; Sacerdot, C.; Tekaia, F.; Leh-Louis, V.; Despons, L.; Khanna, V.; Aury, J.M.; Barbe, V.; Couloux, A.; Labadie, K.; Pelletier, E.; Souciet, J.L.; Boekhout, T.; Gabaldon, T.; Wincker, P.; Dujon, B.

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics, but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid type strain

  12. Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization

    Directory of Open Access Journals (Sweden)

    R. Mezzanotte

    2010-01-01

    Full Text Available The genome of stallion (Spanish breed and donkey (Spanish endemic Zamorano-Leonés were compared using whole comparative genomic in situ hybridization (W-CGH technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

  13. Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization

    Directory of Open Access Journals (Sweden)

    J. Gosalvez

    2010-01-01

    Full Text Available The genome of stallion (Spanish breed and donkey (Spanish endemic Zamorano-Leonés were compared using whole comparative genomic in situ hybridization (W-CGH technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

  14. Isolation and sequence analysis of the wheat B genome subtelomeric DNA

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    Huneau Cecile

    2009-09-01

    Full Text Available Abstract Background Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. Results The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119 737 bp was annotated. It is composed of 33% transposable elements (TEs, 8.2% Spelt52 (namely, the subfamily Spelt52.2 and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11 666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0. Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Conclusion Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat

  15. Genome Sequence Databases (Overview): Sequencing and Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Lapidus, Alla L.

    2009-01-01

    From the date its role in heredity was discovered, DNA has been generating interest among scientists from different fields of knowledge: physicists have studied the three dimensional structure of the DNA molecule, biologists tried to decode the secrets of life hidden within these long molecules, and technologists invent and improve methods of DNA analysis. The analysis of the nucleotide sequence of DNA occupies a special place among the methods developed. Thanks to the variety of sequencing technologies available, the process of decoding the sequence of genomic DNA (or whole genome sequencing) has become robust and inexpensive. Meanwhile the assembly of whole genome sequences remains a challenging task. In addition to the need to assemble millions of DNA fragments of different length (from 35 bp (Solexa) to 800 bp (Sanger)), great interest in analysis of microbial communities (metagenomes) of different complexities raises new problems and pushes some new requirements for sequence assembly tools to the forefront. The genome assembly process can be divided into two steps: draft assembly and assembly improvement (finishing). Despite the fact that automatically performed assembly (or draft assembly) is capable of covering up to 98% of the genome, in most cases, it still contains incorrectly assembled reads. The error rate of the consensus sequence produced at this stage is about 1/2000 bp. A finished genome represents the genome assembly of much higher accuracy (with no gaps or incorrectly assembled areas) and quality ({approx}1 error/10,000 bp), validated through a number of computer and laboratory experiments.

  16. Using physicochemical and compositional characteristics of DNA sequence for prediction of genomic signals

    KAUST Repository

    Mulamba, Pierre Abraham

    2014-12-01

    The challenge in finding genes in eukaryotic organisms using computational methods is an ongoing problem in the biology. Based on various genomic signals found in eukaryotic genomes, this problem can be divided into many different sub­‐problems such as identification of transcription start sites, translation initiation sites, splice sites, poly (A) signals, etc. Each sub-­problem deals with a particular type of genomic signals and various computational methods are used to solve each sub-­problem. Aggregating information from all these individual sub-­problems can lead to a complete annotation of a gene and its component signals. The fundamental principle of most of these computational methods is the mapping principle – building an input-­output model for the prediction of a particular genomic signal based on a set of known input signals and their corresponding output signal. The type of input signals used to build the model is an essential element in most of these computational methods. The common factor of most of these methods is that they are mainly based on the statistical analysis of the basic nucleotide sequence string composition. 4 Our study is based on a novel approach to predict genomic signals in which uniquely generated structural profiles that combine compressed physicochemical properties with topological and compositional properties of DNA sequences are used to develop machine learning predictive models. The compression of the physicochemical properties is made using principal component analysis transformation. Our ideas are evaluated through prediction models of canonical splice sites using support vector machine models. We demonstrate across several species that the proposed methodology has resulted in the most accurate splice site predictors that are publicly available or described. We believe that the approach in this study is quite general and has various applications in other biological modeling problems.

  17. DNA Barcoding: Amplification and sequence analysis of rbcl and matK genome regions in three divergent plant species

    Directory of Open Access Journals (Sweden)

    Javed Iqbal Wattoo

    2016-11-01

    Full Text Available Background: DNA barcoding is a novel method of species identification based on nucleotide diversity of conserved sequences. The establishment and refining of plant DNA barcoding systems is more challenging due to high genetic diversity among different species. Therefore, targeting the conserved nuclear transcribed regions would be more reliable for plant scientists to reveal genetic diversity, species discrimination and phylogeny. Methods: In this study, we amplified and sequenced the chloroplast DNA regions (matk+rbcl of Solanum nigrum, Euphorbia helioscopia and Dalbergia sissoo to study the functional annotation, homology modeling and sequence analysis to allow a more efficient utilization of these sequences among different plant species. These three species represent three families; Solanaceae, Euphorbiaceae and Fabaceae respectively. Biological sequence homology and divergence of amplified sequences was studied using Basic Local Alignment Tool (BLAST. Results: Both primers (matk+rbcl showed good amplification in three species. The sequenced regions reveled conserved genome information for future identification of different medicinal plants belonging to these species. The amplified conserved barcodes revealed different levels of biological homology after sequence analysis. The results clearly showed that the use of these conserved DNA sequences as barcode primers would be an accurate way for species identification and discrimination. Conclusion: The amplification and sequencing of conserved genome regions identified a novel sequence of matK in native species of Solanum nigrum. The findings of the study would be applicable in medicinal industry to establish DNA based identification of different medicinal plant species to monitor adulteration.

  18. Genome sequencing conference II

    Energy Technology Data Exchange (ETDEWEB)

    1990-01-01

    Genome Sequencing Conference 2 was held September 30 to October 30, 1990. 26 speaker abstracts and 33 poster presentations were included in the program report. New and improved methods for DNA sequencing and genetic mapping were presented. Many of the papers were concerned with accuracy and speed of acquisition of data with computers and automation playing an increasing role. Individual papers have been processed separately for inclusion on the database.

  19. Whole genome nucleosome sequencing identifies novel types of forensic markers in degraded DNA samples

    Science.gov (United States)

    Dong, Chun-nan; Yang, Ya-dong; Li, Shu-jin; Yang, Ya-ran; Zhang, Xiao-jing; Fang, Xiang-dong; Yan, Jiang-wei; Cong, Bin

    2016-01-01

    In the case of mass disasters, missing persons and forensic caseworks, highly degraded biological samples are often encountered. It can be a challenge to analyze and interpret the DNA profiles from these samples. Here we provide a new strategy to solve the problem by taking advantage of the intrinsic structural properties of DNA. We have assessed the in vivo positions of more than 35 million putative nucleosome cores in human leukocytes using high-throughput whole genome sequencing, and identified 2,462 single nucleotide variations (SNVs), 128 insertion-deletion polymorphisms (indels). After comparing the sequence reads with 44 STR loci commonly used in forensics, five STRs (TH01, TPOX, D18S51, DYS391, and D10S1248)were matched. We compared these “nucleosome protected STRs” (NPSTRs) with five other non-NPSTRs using mini-STR primer design, real-time PCR, and capillary gel electrophoresis on artificially degraded DNA. Moreover, genotyping performance of the five NPSTRs and five non-NPSTRs was also tested with real casework samples. All results show that loci located in nucleosomes are more likely to be successfully genotyped in degraded samples. In conclusion, after further strict validation, these markers could be incorporated into future forensic and paleontology identification kits, resulting in higher discriminatory power for certain degraded sample types. PMID:27189082

  20. Phylogeny of Pelargonium (Geraniaceae) based on DNA sequences from three genomes

    NARCIS (Netherlands)

    Bakker, F.T.; Culham, A.; Hettiarachi, P.; Touloumendidou, T.; Gibby, M.

    2004-01-01

    Phylogenetic hypotheses for the largely South African genus Pelargonium L'Hér. (Geraniaceae) were derived based on DNA sequence data from nuclear, chloroplast and mitochondrial encoded regions. The datasets were unequally represented and comprised cpDNA trnL-F sequences for 152 taxa, nrDNA ITS seque

  1. Rapid in vitro splicing of coding sequences from genomic DNA by isothermal recombination reaction-based PCR

    Directory of Open Access Journals (Sweden)

    Wenxuan Chen

    2016-09-01

    Full Text Available Cloning of coding sequence (CDS is an important step for gene function research. Here, we reported a simple and efficient strategy for assembling multiple-exon into an intron-free CDS from genomic DNA (gDNA by an isothermal recombination reaction-based PCR (IRR-PCR method. As an example, a 2067-bp full-length CDS of the anther-specific expression gene OsABCG15, which is composed of seven exons and six introns, was generated by IRR-PCR using genomic DNA of rice leaf as the template. Actually, this approach can be wildly applied to any DNA sequences assembly to achieve CDS cloning, gene fusion and multiple site-directed mutagenesis in functional genomics studies in vitro.

  2. Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants.

    Directory of Open Access Journals (Sweden)

    Nadin Rohland

    Full Text Available To elucidate the history of living and extinct elephantids, we generated 39,763 bp of aligned nuclear DNA sequence across 375 loci for African savanna elephant, African forest elephant, Asian elephant, the extinct American mastodon, and the woolly mammoth. Our data establish that the Asian elephant is the closest living relative of the extinct mammoth in the nuclear genome, extending previous findings from mitochondrial DNA analyses. We also find that savanna and forest elephants, which some have argued are the same species, are as or more divergent in the nuclear genome as mammoths and Asian elephants, which are considered to be distinct genera, thus resolving a long-standing debate about the appropriate taxonomic classification of the African elephants. Finally, we document a much larger effective population size in forest elephants compared with the other elephantid taxa, likely reflecting species differences in ancient geographic structure and range and differences in life history traits such as variance in male reproductive success.

  3. Studies of the hyperthermophile Thermotoga maritima by random sequencing of cDNA and genomic libraries. Identification and sequencing of the trpEG (D) operon.

    Science.gov (United States)

    Kim, C W; Markiewicz, P; Lee, J J; Schierle, C F; Miller, J H

    1993-06-20

    Random sequencing of cDNA and genomic libraries has been used to study the genome of the hyperthermophile Thermotoga maritima. To date, 175 unique clones have been analyzed by comparing short sequence tags with known proteins in the PIR and GenBank databases. We find that a significant proportion of sequences can be matched to previously identified protein from non-Thermotoga sources. A high match rate was obtained from an oligo(dT)-primed cDNA library, where one-third of all unique sequences analyzed (21/65) shared high amino acid sequence similarity with proteins in the PIR and GenBank databases. Also, approximately one-third of the unique sequences from a second cDNA library (28/89), constructed with random oligo primers, could be matched to sequences in PIR and GenBank. Identification of genes from the oligo(dT)-primed cDNA library indicates that some Thermotoga mRNAs are polyadenylated. Genes have also been identified from a 1 to 2 kb genomic DNA library. Here, (3/21) of genomic sequences analyzed could be matched to protein in PIR and GenBank. One of the genomic clones had high sequence similarity to the tryptophan synthesis gene anthranilate synthase component I (trpE). Using this sequence tag, the Thermotoga trp operon was isolated and sequenced. The Thermotoga maritima trp operon is arranged with trpE forming an overlapping transcript with a second protein consisting of a fusion of anthranilate synthase component II (trpG) and anthranilate phosphoribosyltransferse (trpD). With regard to the fusion, the operon organization is similar to Escherichia coli and Salmonella typhimurium, but lacks the classic attenuation system of enteric bacteria. Amino acid sequence comparison with 19 trpE, 18 trpG and 14 trpD genes from other organisms suggest that the Thermotoga trp genes resemble corresponding genes from other thermophiles more closely than expected.

  4. SNP discovery using Paired-End RAD-tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae).

    Science.gov (United States)

    Vandepitte, K; Honnay, O; Mergeay, J; Breyne, P; Roldán-Ruiz, I; De Meyer, T

    2013-03-01

    Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost-effective approaches to uncover genome-wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD-PE (Restriction site Associated DNA Paired-End sequencing) approach. RAD tags were generated from the PstI-digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired-end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N(50)  = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD-PE as an inexpensive genome-wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.

  5. Simple and reliable procedure for PCR amplification of genomic DNA from yeast cells using short sequencing primers

    DEFF Research Database (Denmark)

    Haaning, J; Oxvig, C; Overgaard, Michael Toft;

    1997-01-01

    Yeast is widely used in molecular biology. Heterologous expression of recombinant proteins in yeast involves screening of a large number of recombinants. We present an easy and reliable procedure for amplifying genomic DNA from freshly grown cells of the methylotrophic yeast Pichia pastoris...... by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature...

  6. DNA sequencing by CE.

    Science.gov (United States)

    Karger, Barry L; Guttman, András

    2009-06-01

    Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA-sequencing methods have evolved from the labor-intensive slab gel electrophoresis, through automated multiCE systems using fluorophore labeling with multispectral imaging, to the "next-generation" technologies of cyclic-array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes were only possible with the advent of modern sequencing technologies that were a result of step-by-step advances with a contribution of academics, medical personnel and instrument companies. While next-generation sequencing is moving ahead at breakneck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of CE in DNA sequencing based in part of several of our articles in this journal.

  7. Long span DNA paired-end-tag (DNA-PET sequencing strategy for the interrogation of genomic structural mutations and fusion-point-guided reconstruction of amplicons.

    Directory of Open Access Journals (Sweden)

    Fei Yao

    Full Text Available Structural variations (SVs contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET sequencing data using large DNA inserts of 10-20 kb and compared their characteristics with short insert (1 kb libraries for their ability to identify SVs. Our results suggest that although short insert libraries bear an advantage in identifying small deletions, they do not provide significantly better breakpoint resolution. In contrast, large inserts are superior to short inserts in providing higher physical genome coverage for the same sequencing cost and achieve greater sensitivity, in practice, for the identification of several classes of SVs, such as copy number neutral and complex events. Furthermore, our results confirm that large insert libraries allow for the identification of SVs within repetitive sequences, which cannot be spanned by short inserts. This provides a key advantage in studying rearrangements in cancer, and we show how it can be used in a fusion-point-guided-concatenation algorithm to study focally amplified regions in cancer.

  8. The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

    Directory of Open Access Journals (Sweden)

    Olivier Arnaiz

    Full Text Available Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a

  9. The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

    Science.gov (United States)

    Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

    2012-01-01

    Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the

  10. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave; Lescai, Francesco; Grove, Jakob;

    2016-01-01

    and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference--whole-blood DNA--based on concordance rates calculated...

  11. Co-barcoded sequence reads from long DNA fragments: A cost-effective solution for Perfect Genome sequencing

    Directory of Open Access Journals (Sweden)

    Brock A Peters

    2015-01-01

    Full Text Available Next generation sequencing (NGS technologies, primarily based on massively parallel sequencing (MPS, have touched and radically changed almost all aspects of research worldwide. These technologies have allowed for the rapid analysis, to date, of the genomes of more than 2,000 different species. In humans, NGS has arguably had the largest impact. Over 100,000 genomes of individual humans (based on various estimates have been sequenced allowing for deep insights into what makes individuals and families unique and what causes disease in each of us. Despite all of this progress, the current state of the art in sequence technology is far from generating a perfect genome sequence and much remains to be understood in the biology of human and other organisms’ genomes. In the article that follows we outline, why the perfect genome in humans is important, what is lacking from current human whole genome sequences, and a potential strategy for achieving the perfect genome in a cost effective manner.

  12. Selection of Unique Escherichia coli Clones by Random Amplified Polymorphic DNA (RAPD): Evaluation by Whole Genome Sequencing

    Science.gov (United States)

    Nielsen, Karen L.; Godfrey, Paul A.; Stegger, Marc; Andersen, Paal S.; Feldgarden, Michael; Frimodt-Møller, Niels

    2014-01-01

    Identifying and characterizing clonal diversity is important when analysing fecal flora. We evaluated random amplified polymorphic DNA (RAPD) PCR, applied for selection of Escherichia coli isolates, by whole genome sequencing. RAPD was fast, and reproducible as screening method for selection of distinct E. coli clones in fecal swabs. PMID:24912108

  13. Whole genome semiconductor based sequencing of farmed European sea bass (Dicentrarchus labrax) Mediterranean genetic stocks using a DNA pooling approach.

    Science.gov (United States)

    Bertolini, Francesca; Geraci, Claudia; Schiavo, Giuseppina; Sardina, Maria Teresa; Chiofalo, Vincenzo; Fontanesi, Luca

    2016-08-01

    European sea bass (Dicentrarchus labrax) is an important marine species for commercial and sport fisheries and aquaculture production. Recently, the European sea bass genome has been sequenced and assembled. This resource can open new opportunities to evaluate and monitor variability and identify variants that could contribute to the adaptation to farming conditions. In this work, two DNA pools constructed from cultivated European sea bass were sequenced using a next generation semiconductor sequencing approach based on Ion Proton sequencer. Using the first draft version of the D. labrax genome as reference, sequenced reads obtained a total of about 1.6 million of single nucleotide polymorphisms (SNPs), spread all over the chromosomes. Transition/transversion (Ti/Tv) was equal to 1.28, comparable to what was already reported in Salmon species. A pilot homozygosity analysis across the D. labrax genome using DNA pool sequence datasets indicated that this approach can identify chromosome regions with putative signatures of selection, including genes involved in ion transport and chloride channel functions, amino acid metabolism and circadian clock and related neurological systems. This is the first study that reported genome wide polymorphisms in a fish species obtained with the Ion Proton sequencer. Moreover, this study provided a methodological approach for selective sweep analysis in this species.

  14. Measurement of word frequencies in genomic DNA sequences based on partial alignment and fuzzy set.

    Science.gov (United States)

    Shida, Fumiya; Mizuta, Satoshi

    2014-08-01

    Accompanied with the rapid increase of the amount of data registered in the databases of biological sequences, the need for a fast method of sequence comparison applicable to sequences of large size is also increasing. In general, alignment is used for sequence comparison. However, the alignment may not be appropriate for comparison of sequences of large size such as whole genome sequences due to its large time complexity. In this article, we propose a semi alignment-free method of sequence comparison based on word frequency distributions, in which we partially use the alignment to measure word frequencies along with the idea of fuzzy set theory. Experiments with ten bacterial genome sequences demonstrated that the fuzzy measurements has the effect that facilitates discrimination between close relatives and distant relatives.

  15. Complete nucleotide sequences of the domestic cat (Felis catus) mitochondrial genome and a transposed mtDNA tandem repeat (Numt) in the nuclear genome

    Energy Technology Data Exchange (ETDEWEB)

    Lopez, J.V.; Cevario, S.; O`Brien, S.J. [National Cancer Institute, Frederick, MD (United States)

    1996-04-15

    The complete 17,009-bp mitochondrial genome of the domestic cat, Felis catus, has been sequenced and conforms largely to the typical organization of previously characterized mammalian mtDNAs. Codon usage and base composition also followed canonical vertebrate patterns, except for an unusual ATC (non-AUG) codon initiating the NADH dehydrogenase subunit 2 (ND2) gene. Two distinct repetitive motifs at opposite ends of the control region contribute to the relatively large size (1559 bp) of this carnivore mtDNA. Alignment of the feline mtDNA genome to a homologous 7946-bp nuclear mtDNA tandem repeat DNA sequence in the cat, Numt, indicates simple repeat motifs associated with insertion/deletion mutations. Overall DNA sequence divergence between Numt and cytoplasmic mtDNA sequence was only 5.1%. Substitutions predominate at the third codon position of homologous feline protein genes. Phylogenetic analysis of mitochondrial gene sequences confirms the recent transfer of the cytoplasmic mtDNA sequences to the domestic cat nucleus and recapitulates evolutionary relationships between mammal species. 86 refs., 4 figs., 3 tabs.

  16. The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity.

    Science.gov (United States)

    Dapprich, Johannes; Ferriola, Deborah; Mackiewicz, Kate; Clark, Peter M; Rappaport, Eric; D'Arcy, Monica; Sasson, Ariella; Gai, Xiaowu; Schug, Jonathan; Kaestner, Klaus H; Monos, Dimitri

    2016-07-09

    The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3'-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing

  17. The Complete DNA Sequence of the Mitochondrial Genome of a ``living Fossil,'' the Coelacanth (Latimeria Chalumnae)

    OpenAIRE

    Zardoya, Rafael; Meyer, Axel

    1997-01-01

    The complete nucleotide sequence of the 16,407-bp mitochondrial genome of the coelacanth (Latirneria chalumnae) was determined. Thec oelacanth mitochondrial genome orderi s identical to the consensus vertebrate gene order which is also found in all ray-finned fishes, the lungfish, and most tetrapods. Base composition and codon usage also conform to typical vertebrate patterns. The entire mitochondrial genome was PCR-amplified with 24 sets of primers that are expected to amplify homologous reg...

  18. Determination of the melon chloroplast and mitochondrial genome sequences reveals that the largest reported mitochondrial genome in plants contains a significant amount of DNA having a nuclear origin

    Science.gov (United States)

    2011-01-01

    Background The melon belongs to the Cucurbitaceae family, whose economic importance among vegetable crops is second only to Solanaceae. The melon has a small genome size (454 Mb), which makes it suitable for molecular and genetic studies. Despite similar nuclear and chloroplast genome sizes, cucurbits show great variation when their mitochondrial genomes are compared. The melon possesses the largest plant mitochondrial genome, as much as eight times larger than that of other cucurbits. Results The nucleotide sequences of the melon chloroplast and mitochondrial genomes were determined. The chloroplast genome (156,017 bp) included 132 genes, with 98 single-copy genes dispersed between the small (SSC) and large (LSC) single-copy regions and 17 duplicated genes in the inverted repeat regions (IRa and IRb). A comparison of the cucumber and melon chloroplast genomes showed differences in only approximately 5% of nucleotides, mainly due to short indels and SNPs. Additionally, 2.74 Mb of mitochondrial sequence, accounting for 95% of the estimated mitochondrial genome size, were assembled into five scaffolds and four additional unscaffolded contigs. An 84% of the mitochondrial genome is contained in a single scaffold. The gene-coding region accounted for 1.7% (45,926 bp) of the total sequence, including 51 protein-coding genes, 4 conserved ORFs, 3 rRNA genes and 24 tRNA genes. Despite the differences observed in the mitochondrial genome sizes of cucurbit species, Citrullus lanatus (379 kb), Cucurbita pepo (983 kb) and Cucumis melo (2,740 kb) share 120 kb of sequence, including the predicted protein-coding regions. Nevertheless, melon contained a high number of repetitive sequences and a high content of DNA of nuclear origin, which represented 42% and 47% of the total sequence, respectively. Conclusions Whereas the size and gene organisation of chloroplast genomes are similar among the cucurbit species, mitochondrial genomes show a wide variety of sizes, with a non

  19. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    2001-01-01

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus whi

  20. Sequence space coverage, entropy of genomes and the potential to detect non-human DNA in human samples

    Directory of Open Access Journals (Sweden)

    Maley Carlo C

    2008-10-01

    Full Text Available Abstract Background Genomes store information for building and maintaining organisms. Complete sequencing of many genomes provides the opportunity to study and compare global information properties of those genomes. Results We have analyzed aspects of the information content of Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli (K-12 genomes. Virtually all possible (> 98% 12 bp oligomers appear in vertebrate genomes while 98% to D. melanogaster (12–17 bp, C. elegans (11–17 bp, A. thaliana (11–17 bp, S. cerevisiae (10–16 bp and E. coli (9–15 bp. Frequencies of unique oligomers in the genomes follow similar patterns. We identified a set of 2.6 M 15-mers that are more than 1 nucleotide different from all 15-mers in the human genome and so could be used as probes to detect microbes in human samples. In a human sample, these probes would detect 100% of the 433 currently fully sequenced prokaryotes and 75% of the 3065 fully sequenced viruses. The human genome is significantly more compact in sequence space than a random genome. We identified the most frequent 5- to 20-mers in the human genome, which may prove useful as PCR primers. We also identified a bacterium, Anaeromyxobacter dehalogenans, which has an exceptionally low diversity of oligomers given the size of its genome and its GC content. The entropy of coding regions in the human genome is significantly higher than non-coding regions and chromosomes. However chromosomes 1, 2, 9, 12 and 14 have a relatively high proportion of coding DNA without high entropy, and chromosome 20 is the opposite with a low frequency of coding regions but relatively high entropy. Conclusion Measures of the frequency of oligomers are useful for designing PCR assays and for identifying chromosomes and organisms with hidden structure that had not been previously recognized. This information may be used to detect

  1. DNA Lossless Differential Compression Algorithm based on Similarity of Genomic Sequence Database

    CERN Document Server

    Afify, Heba; Wahed, Manal Abdel

    2011-01-01

    Modern biological science produces vast amounts of genomic sequence data. This is fuelling the need for efficient algorithms for sequence compression and analysis. Data compression and the associated techniques coming from information theory are often perceived as being of interest for data communication and storage. In recent years, a substantial effort has been made for the application of textual data compression techniques to various computational biology tasks, ranging from storage and indexing of large datasets to comparison of genomic databases. This paper presents a differential compression algorithm that is based on production of difference sequences according to op-code table in order to optimize the compression of homologous sequences in dataset. Therefore, the stored data are composed of reference sequence, the set of differences, and differences locations, instead of storing each sequence individually. This algorithm does not require a priori knowledge about the statistics of the sequence set. The...

  2. Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing.

    Directory of Open Access Journals (Sweden)

    Sumitra Mohan

    2014-03-01

    Full Text Available Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR, such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC. However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%, which occurred either as novel focal amplifications (n = 3 or as high level polysomy of 12p (n = 1. In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2 and ERBB2 (n = 1. Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%, which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

  3. Proceedings of the relevance of mass spectrometry to DNA sequence determination: Research needs for the Human Genome Program

    Energy Technology Data Exchange (ETDEWEB)

    Edmonds, C.G.; Smith, R.D. (Pacific Northwest Lab., Richland, WA (USA)); Smith, L.M. (Wisconsin Univ., Madison, WI (USA))

    1990-11-01

    A workshop was sponsored for the US Department of Energy (DOE), Office of Health and Environmental Research by Pacific Northwest Laboratory, April 4--5, 1990, in Seattle, Washington, to examine the potential role of mass spectrometry in the joint DOE/National Institutes of Health (NIH) Human Genome Program. The workshop was occasioned by recent developments in mass spectrometry that are providing new levels for selectivity, sensitivity, and, in particular, new methods of ionization appropriate for large biopolymers such as DNA. During discussions, three general mass spectrometric approaches to the determination of DNA sequence were considered: (1) the mass spectrometric detection of isotopic labels from DNA sequencing mixtures separated using gel electrophoresis, (2) the direct mass spectrometric analysis from direct ionization of unfractionated sequencing mixtures where the measured mass of the constituents functions to identify and order the base sequence (replacing separation by gel electrophoresis), and (3) an approach in which a single highly charged molecular ion of a large DNA segment produced is rapidly sequenced in an ion cyclotron resonance ion trap. The consensus of the workshop was that, on the basis of the new developments, mass spectrometry has the potential to provide the substantial increases in sequencing speed required for the Human Genome Program. 66 refs., 3 tabs.

  4. Proceedings of the relevance of mass spectrometry to DNA sequence determination: Research needs for the Human Genome Program

    Energy Technology Data Exchange (ETDEWEB)

    Edmonds, C.G.; Smith, R.D. (Pacific Northwest Lab., Richland, WA (USA)); Smith, L.M. (Wisconsin Univ., Madison, WI (USA))

    1990-11-01

    A workshop was sponsored for the US Department of Energy (DOE), Office of Health and Environmental Research by Pacific Northwest Laboratory, April 4--5, 1990, in Seattle, Washington, to examine the potential role of mass spectrometry in the joint DOE/National Institutes of Health (NIH) Human Genome Program. The workshop was occasioned by recent developments in mass spectrometry that are providing new levels for selectivity, sensitivity, and, in particular, new methods of ionization appropriate for large biopolymers such as DNA. During discussions, three general mass spectrometric approaches to the determination of DNA sequence were considered: (1) the mass spectrometric detection of isotopic labels from DNA sequencing mixtures separated using gel electrophoresis, (2) the direct mass spectrometric analysis from direct ionization of unfractionated sequencing mixtures where the measured mass of the constituents functions to identify and order the base sequence (replacing separation by gel electrophoresis), and (3) an approach in which a single highly charged molecular ion of a large DNA segment produced is rapidly sequenced in an ion cyclotron resonance ion trap. The consensus of the workshop was that, on the basis of the new developments, mass spectrometry has the potential to provide the substantial increases in sequencing speed required for the Human Genome Program. 66 refs., 3 tabs.

  5. Promoter prediction and annotation of microbial genomes based on DNA sequence and structural responses to superhelical stress

    Directory of Open Access Journals (Sweden)

    Benham Craig J

    2006-05-01

    Full Text Available Abstract Background In our previous studies, we found that the sites in prokaryotic genomes which are most susceptible to duplex destabilization under the negative superhelical stresses that occur in vivo are statistically highly significantly associated with intergenic regions that are known or inferred to contain promoters. In this report we investigate how this structural property, either alone or together with other structural and sequence attributes, may be used to search prokaryotic genomes for promoters. Results We show that the propensity for stress-induced DNA duplex destabilization (SIDD is closely associated with specific promoter regions. The extent of destabilization in promoter-containing regions is found to be bimodally distributed. When compared with DNA curvature, deformability, thermostability or sequence motif scores within the -10 region, SIDD is found to be the most informative DNA property regarding promoter locations in the E. coli K12 genome. SIDD properties alone perform better at detecting promoter regions than other programs trained on this genome. Because this approach has a very low false positive rate, it can be used to predict with high confidence the subset of promoters that are strongly destabilized. When SIDD properties are combined with -10 motif scores in a linear classification function, they predict promoter regions with better than 80% accuracy. When these methods were tested with promoter and non-promoter sequences from Bacillus subtilis, they achieved similar or higher accuracies. We also present a strictly SIDD-based predictor for annotating promoter sequences in complete microbial genomes. Conclusion In this report we show that the propensity to undergo stress-induced duplex destabilization (SIDD is a distinctive structural attribute of many prokaryotic promoter sequences. We have developed methods to identify promoter sequences in prokaryotic genomes that use SIDD either as a sole predictor or in

  6. Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

    DEFF Research Database (Denmark)

    Winkel, Bo G; Hollegaard, Mads Vilhelm; Olesen, Morten S;

    2011-01-01

    The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare m...

  7. Identification and annotation of promoter regions in microbial genome sequences on the basis of DNA stability

    Indian Academy of Sciences (India)

    Vetriselvi Rangannan; Manju Bansal

    2007-08-01

    Analysis of various predicted structural properties of promoter regions in prokaryotic as well as eukaryotic genomes had earlier indicated that they have several common features, such as lower stability, higher curvature and less bendability, when compared with their neighboring regions. Based on the difference in stability between neighboring upstream and downstream regions in the vicinity of experimentally determined transcription start sites, a promoter prediction algorithm has been developed to identify prokaryotic promoter sequences in whole genomes. The average free energy (E) over known promoter sequences and the difference (D) between E and the average free energy over the entire genome (G) are used to search for promoters in the genomic sequences. Using these cutoff values to predict promoter regions across entire Escherichia coli genome, we achieved a reliability of 70% when the predicted promoters were cross verified against the 960 transcription start sites (TSSs) listed in the Ecocyc database. Annotation of the whole E. coli genome for promoter region could be carried out with 49% accuracy. The method is quite general and it can be used to annotate the promoter regions of other prokaryotic genomes.

  8. 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.

  9. Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: an ultra-large metazoan mitochondrial genome.

    Science.gov (United States)

    Liu, Yun-Guo; Kurokawa, Tadahide; Sekino, Masashi; Tanabe, Toru; Watanabe, Kazuhito

    2013-03-01

    The complete mitochondrial (mt) genome of the ark shell Scapharca broughtonii was determined using long PCR and a genome walking sequencing strategy with genus-specific primers. The S. broughtonii mt genome (GenBank accession number AB729113) contained 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 2 ribosomal RNA genes, and 42 transfer tRNA genes, in a length of 46,985 nucleotides for the size of mtDNA with only one copy of the heteroplasmic tandem repeat (HTR) unit. Moreover the S. broughtonii mt genome shows size variation; these genomes ranged in size from about 47 kb to about 50 kb because of variation in the number of repeat sequences in the non-coding region. The mt-genome of S. broughtonii is, to date, the longest reported metazoan mtDNA sequence. Sequence duplication in non-coding region and the formation of HTR arrays were two of the factors responsible for the ultra-large size of this mt genome. All the tRNA genes were found within the S. broughtonii mt genome, unlike the other bivalves usually lacking one or more tRNA genes. Twelve additional specimens were used to analyze the patterns of tandem repeat arrays by PCR amplification and agarose electrophoresis. Each of the 12 specimens displayed extensive heteroplasmy and had 8-10 length variants. The motifs of the HTR arrays are about 353-362 bp and the number of repeats ranges from 1 to 11.

  10. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

    Science.gov (United States)

    Hykin, Sarah M.; Bi, Ke; McGuire, Jimmy A.

    2015-01-01

    For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

  11. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    Science.gov (United States)

    Hykin, Sarah M; Bi, Ke; McGuire, Jimmy A

    2015-01-01

    For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

  12. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    Directory of Open Access Journals (Sweden)

    Sarah M Hykin

    Full Text Available For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles, attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp. We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens

  13. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.

    Science.gov (United States)

    McKenna, Aaron; Hanna, Matthew; Banks, Eric; Sivachenko, Andrey; Cibulskis, Kristian; Kernytsky, Andrew; Garimella, Kiran; Altshuler, David; Gabriel, Stacey; Daly, Mark; DePristo, Mark A

    2010-09-01

    Next-generation DNA sequencing (NGS) projects, such as the 1000 Genomes Project, are already revolutionizing our understanding of genetic variation among individuals. However, the massive data sets generated by NGS--the 1000 Genome pilot alone includes nearly five terabases--make writing feature-rich, efficient, and robust analysis tools difficult for even computationally sophisticated individuals. Indeed, many professionals are limited in the scope and the ease with which they can answer scientific questions by the complexity of accessing and manipulating the data produced by these machines. Here, we discuss our Genome Analysis Toolkit (GATK), a structured programming framework designed to ease the development of efficient and robust analysis tools for next-generation DNA sequencers using the functional programming philosophy of MapReduce. The GATK provides a small but rich set of data access patterns that encompass the majority of analysis tool needs. Separating specific analysis calculations from common data management infrastructure enables us to optimize the GATK framework for correctness, stability, and CPU and memory efficiency and to enable distributed and shared memory parallelization. We highlight the capabilities of the GATK by describing the implementation and application of robust, scale-tolerant tools like coverage calculators and single nucleotide polymorphism (SNP) calling. We conclude that the GATK programming framework enables developers and analysts to quickly and easily write efficient and robust NGS tools, many of which have already been incorporated into large-scale sequencing projects like the 1000 Genomes Project and The Cancer Genome Atlas.

  14. Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

    Science.gov (United States)

    Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

    2012-05-01

    The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.

  15. cDNA, genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda

    Science.gov (United States)

    Hou, Yi-ling; Hou, Wan-ru; Ren, Zheng-long; Hao, Yan-zhe; Zhang, Tian

    2008-01-01

    αB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further. PMID:19043608

  16. Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

    DEFF Research Database (Denmark)

    Marie, Rodolphe; Pedersen, Jonas Nyvold; L. V. Bauer, David

    2013-01-01

    We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratoryon-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known...

  17. Genomic Analysis of a Marine Bacterium: Bioinformatics for Comparison, Evaluation, and Interpretation of DNA Sequences

    Directory of Open Access Journals (Sweden)

    Bhagwan N. Rekadwad

    2016-01-01

    Full Text Available A total of five highly related strains of an unidentified marine bacterium were analyzed through their short genome sequences (AM260709–AM260713. Genome-to-Genome Distance (GGDC showed high similarity to Pseudoalteromonas haloplanktis (X67024. The generated unique Quick Response (QR codes indicated no identity to other microbial species or gene sequences. Chaos Game Representation (CGR showed the number of bases concentrated in the area. Guanine residues were highest in number followed by cytosine. Frequency of Chaos Game Representation (FCGR indicated that CC and GG blocks have higher frequency in the sequence from the evaluated marine bacterium strains. Maximum GC content for the marine bacterium strains ranged 53-54%. The use of QR codes, CGR, FCGR, and GC dataset helped in identifying and interpreting short genome sequences from specific isolates. A phylogenetic tree was constructed with the bootstrap test (1000 replicates using MEGA6 software. Principal Component Analysis (PCA was carried out using EMBL-EBI MUSCLE program. Thus, generated genomic data are of great assistance for hierarchical classification in Bacterial Systematics which combined with phenotypic features represents a basic procedure for a polyphasic approach on unambiguous bacterial isolate taxonomic classification.

  18. Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    Directory of Open Access Journals (Sweden)

    Stougaard Magnus

    2007-11-01

    Full Text Available Abstract Background In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. Methods Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. Results An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1 on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. Conclusion Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as

  19. Complete DNA sequence of Kuraishia capsulata illustrates novel genomic features among budding yeasts (Saccharomycotina).

    Science.gov (United States)

    Morales, Lucia; Noel, Benjamin; Porcel, Betina; Marcet-Houben, Marina; Hullo, Marie-Francoise; Sacerdot, Christine; Tekaia, Fredj; Leh-Louis, Véronique; Despons, Laurence; Khanna, Varun; Aury, Jean-Marc; Barbe, Valérie; Couloux, Arnaud; Labadie, Karen; Pelletier, Eric; Souciet, Jean-Luc; Boekhout, Teun; Gabaldon, Toni; Wincker, Patrick; Dujon, Bernard

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993(T)), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ~13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts.

  20. Complete DNA Sequence of Kuraishia capsulata Illustrates Novel Genomic Features among Budding Yeasts (Saccharomycotina)

    Science.gov (United States)

    Morales, Lucia; Noel, Benjamin; Porcel, Betina; Marcet-Houben, Marina; Hullo, Marie-Francoise; Sacerdot, Christine; Tekaia, Fredj; Leh-Louis, Véronique; Despons, Laurence; Khanna, Varun; Aury, Jean-Marc; Barbe, Valérie; Couloux, Arnaud; Labadie, Karen; Pelletier, Eric; Souciet, Jean-Luc; Boekhout, Teun; Gabaldon, Toni; Wincker, Patrick; Dujon, Bernard

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993T), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ∼13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts. PMID:24317973

  1. Structural variation in the chicken genome identified by paired-end next-generation DNA sequencing of reduced representation libraries

    Directory of Open Access Journals (Sweden)

    Okimoto Ron

    2011-02-01

    Full Text Available Abstract Background Variation within individual genomes ranges from single nucleotide polymorphisms (SNPs to kilobase, and even megabase, sized structural variants (SVs, such as deletions, insertions, inversions, and more complex rearrangements. Although much is known about the extent of SVs in humans and mice, species in which they exert significant effects on phenotypes, very little is known about the extent of SVs in the 2.5-times smaller and less repetitive genome of the chicken. Results We identified hundreds of shared and divergent SVs in four commercial chicken lines relative to the reference chicken genome. The majority of SVs were found in intronic and intergenic regions, and we also found SVs in the coding regions. To identify the SVs, we combined high-throughput short read paired-end sequencing of genomic reduced representation libraries (RRLs of pooled samples from 25 individuals and computational mapping of DNA sequences from a reference genome. Conclusion We provide a first glimpse of the high abundance of small structural genomic variations in the chicken. Extrapolating our results, we estimate that there are thousands of rearrangements in the chicken genome, the majority of which are located in non-coding regions. We observed that structural variation contributes to genetic differentiation among current domesticated chicken breeds and the Red Jungle Fowl. We expect that, because of their high abundance, SVs might explain phenotypic differences and play a role in the evolution of the chicken genome. Finally, our study exemplifies an efficient and cost-effective approach for identifying structural variation in sequenced genomes.

  2. Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage

    Directory of Open Access Journals (Sweden)

    Maugel Timothy K

    2007-07-01

    Full Text Available Abstract Background Bioinformatic analysis of the genome sequence of Neisseria gonorrhoeae revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown. Results Our analysis of the genomic sequence of FA1090 identified five genomic regions (NgoΦ1 – 5 that are related to dsDNA lysogenic phage. The genetic content of the dsDNA prophage sequences were examined in detail and found to contain blocks of genes encoding for proteins homologous to proteins responsible for phage DNA replication, structural proteins and proteins responsible for phage assembly. The DNA sequences from NgoΦ1, NgoΦ2 and NgoΦ3 contain some significant regions of identity. A unique region of NgoΦ2 showed very high similarity with the Pseudomonas aeruginosa generalized transducing phage F116. Comparative analysis at the nucleotide and protein levels suggests that the sequences of NgoΦ1 and NgoΦ2 encode functionally active phages, while NgoΦ3, NgoΦ4 and NgoΦ5 encode incomplete genomes. Expression of the NgoΦ1 and NgoΦ2 repressors in Escherichia coli inhibit the growth of E. coli and the propagation of phage λ. The NgoΦ2 repressor was able to inhibit transcription of N. gonorrhoeae genes and Haemophilus influenzae HP1 phage promoters. The holin gene of NgoΦ1 (identical to that encoded by NgoΦ2, when expressed in E. coli, could serve as substitute for the phage λ s gene. We were able to detect the presence of the DNA derived from NgoΦ1 in the cultures of N. gonorrhoeae. Electron microscopy analysis of culture supernatants revealed the presence of multiple forms of bacteriophage particles. Conclusion These data suggest that the genes similar to dsDNA lysogenic phage present in the gonococcus are generally conserved in this pathogen and that they are able to regulate the expression of other neisserial genes. Since phage particles were

  3. Extensive sequence-influenced DNA methylation polymorphism in the human genome

    Directory of Open Access Journals (Sweden)

    Hellman Asaf

    2010-05-01

    Full Text Available Abstract Background Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters, but there are also interesting patterns of CpG methylation found outside of CpG islands. Results We compared DNA methylation patterns on both alleles between many pairs (and larger groups of related and unrelated individuals. Direct observation and simulation experiments revealed that around 10% of common single nucleotide polymorphisms (SNPs reside in regions with differences in the propensity for local DNA methylation between the two alleles. We further showed that for the most common form of SNP, a polymorphism at a CpG dinucleotide, the presence of the CpG at the SNP positively affected local DNA methylation in cis. Conclusions Taken together with the known effect of DNA methylation on mutation rate, our results suggest an interesting interdependence between genetics and epigenetics underlying diversity in the human genome.

  4. Whole-exome/genome sequencing and genomics.

    Science.gov (United States)

    Grody, Wayne W; Thompson, Barry H; Hudgins, Louanne

    2013-12-01

    As medical genetics has progressed from a descriptive entity to one focused on the functional relationship between genes and clinical disorders, emphasis has been placed on genomics. Genomics, a subelement of genetics, is the study of the genome, the sum total of all the genes of an organism. The human genome, which is contained in the 23 pairs of nuclear chromosomes and in the mitochondrial DNA of each cell, comprises >6 billion nucleotides of genetic code. There are some 23,000 protein-coding genes, a surprisingly small fraction of the total genetic material, with the remainder composed of noncoding DNA, regulatory sequences, and introns. The Human Genome Project, launched in 1990, produced a draft of the genome in 2001 and then a finished sequence in 2003, on the 50th anniversary of the initial publication of Watson and Crick's paper on the double-helical structure of DNA. Since then, this mass of genetic information has been translated at an ever-increasing pace into useable knowledge applicable to clinical medicine. The recent advent of massively parallel DNA sequencing (also known as shotgun, high-throughput, and next-generation sequencing) has brought whole-genome analysis into the clinic for the first time, and most of the current applications are directed at children with congenital conditions that are undiagnosable by using standard genetic tests for single-gene disorders. Thus, pediatricians must become familiar with this technology, what it can and cannot offer, and its technical and ethical challenges. Here, we address the concepts of human genomic analysis and its clinical applicability for primary care providers.

  5. Genomic DNA pooling strategy for next-generation sequencing-based rare variant discovery in abdominal aortic aneurysm regions of interest-challenges and limitations

    NARCIS (Netherlands)

    Harakalova, M.; Nijman, I.J.; Medic, J.; Mokry, M.; Renkens, I.; Blankensteijn, J.D.; Kloosterman, W.P.; Baas, A.F.; Cuppen, E.

    2011-01-01

    The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA

  6. PipMaker—A Web Server for Aligning Two Genomic DNA Sequences

    OpenAIRE

    Schwartz, Scott; Zheng ZHANG; Frazer, Kelly A; Smit, Arian; Riemer, Cathy; Bouck, John; Gibbs, Richard; Hardison, Ross; Miller, Webb

    2000-01-01

    PipMaker (http://bio.cse.psu.edu) is a World-Wide Web site for comparing two long DNA sequences to identify conserved segments and for producing informative, high-resolution displays of the resulting alignments. One display is a percent identity plot (pip), which shows both the position in one sequence and the degree of similarity for each aligning segment between the two sequences in a compact and easily understandable form. Positions along the horizontal axis can be labeled with features su...

  7. Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations

    DEFF Research Database (Denmark)

    Rausch, Tobias; Jones, David T W; Zapatka, Marc

    2012-01-01

    of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals...

  8. Reduced representation bisulphite sequencing of the cattle genome reveals DNA methylation patterns

    Science.gov (United States)

    Using reduced representation bisulphite sequencing (RRBS), we obtained the first single-base-resolution maps of bovine DNA methylation in ten somatic tissues. In total, we observed 1,868,049 cytosines in the CG-enriched regions. Similar to the methylation patterns in other species, the CG context wa...

  9. Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry ( Prunus avium L.).

    Science.gov (United States)

    Wünsch, A; Hormaza, J I

    2004-01-01

    Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease ( S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5' flanking regions of four S-RNases of sweet cherry ( Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S23, S24 and S25 present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S12 amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5'-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5'-flanking regions of S-RNases from the Maloideae. S6- and S24-RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.

  10. Genome-wide footprints of pig domestication and selection revealed through massive parallel sequencing of pooled DNA.

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    Andreia J Amaral

    Full Text Available BACKGROUND: Artificial selection has caused rapid evolution in domesticated species. The identification of selection footprints across domesticated genomes can contribute to uncover the genetic basis of phenotypic diversity. METHODOLOGY/MAIN FINDINGS: Genome wide footprints of pig domestication and selection were identified using massive parallel sequencing of pooled reduced representation libraries (RRL representing ∼2% of the genome from wild boar and four domestic pig breeds (Large White, Landrace, Duroc and Pietrain which have been under strong selection for muscle development, growth, behavior and coat color. Using specifically developed statistical methods that account for DNA pooling, low mean sequencing depth, and sequencing errors, we provide genome-wide estimates of nucleotide diversity and genetic differentiation in pig. Widespread signals suggestive of positive and balancing selection were found and the strongest signals were observed in Pietrain, one of the breeds most intensively selected for muscle development. Most signals were population-specific but affected genomic regions which harbored genes for common biological categories including coat color, brain development, muscle development, growth, metabolism, olfaction and immunity. Genetic differentiation in regions harboring genes related to muscle development and growth was higher between breeds than between a given breed and the wild boar. CONCLUSIONS/SIGNIFICANCE: These results, suggest that although domesticated breeds have experienced similar selective pressures, selection has acted upon different genes. This might reflect the multiple domestication events of European breeds or could be the result of subsequent introgression of Asian alleles. Overall, it was estimated that approximately 7% of the porcine genome has been affected by selection events. This study illustrates that the massive parallel sequencing of genomic pools is a cost-effective approach to identify

  11. Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing.

    Directory of Open Access Journals (Sweden)

    Jeong-Hyeon Choi

    Full Text Available BACKGROUND: Follicular lymphoma (FL is a form of non-Hodgkin's lymphoma (NHL that arises from germinal center (GC B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

  12. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

    Directory of Open Access Journals (Sweden)

    Bharti Arvind K

    2008-12-01

    Full Text Available Abstract Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR and methylation spanning linker libraries (MSLL. These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig, while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%. These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of

  13. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing.

    Directory of Open Access Journals (Sweden)

    Eric Talevich

    2016-04-01

    Full Text Available Germline copy number variants (CNVs and somatic copy number alterations (SCNAs are of significant importance in syndromic conditions and cancer. Massively parallel sequencing is increasingly used to infer copy number information from variations in the read depth in sequencing data. However, this approach has limitations in the case of targeted re-sequencing, which leaves gaps in coverage between the regions chosen for enrichment and introduces biases related to the efficiency of target capture and library preparation. We present a method for copy number detection, implemented in the software package CNVkit, that uses both the targeted reads and the nonspecifically captured off-target reads to infer copy number evenly across the genome. This combination achieves both exon-level resolution in targeted regions and sufficient resolution in the larger intronic and intergenic regions to identify copy number changes. In particular, we successfully inferred copy number at equivalent to 100-kilobase resolution genome-wide from a platform targeting as few as 293 genes. After normalizing read counts to a pooled reference, we evaluated and corrected for three sources of bias that explain most of the extraneous variability in the sequencing read depth: GC content, target footprint size and spacing, and repetitive sequences. We compared the performance of CNVkit to copy number changes identified by array comparative genomic hybridization. We packaged the components of CNVkit so that it is straightforward to use and provides visualizations, detailed reporting of significant features, and export options for integration into existing analysis pipelines. CNVkit is freely available from https://github.com/etal/cnvkit.

  14. DNA Data Visualization (DDV): Software for Generating Web-Based Interfaces Supporting Navigation and Analysis of DNA Sequence Data of Entire Genomes.

    Science.gov (United States)

    Neugebauer, Tomasz; Bordeleau, Eric; Burrus, Vincent; Brzezinski, Ryszard

    2015-01-01

    Data visualization methods are necessary during the exploration and analysis activities of an increasingly data-intensive scientific process. There are few existing visualization methods for raw nucleotide sequences of a whole genome or chromosome. Software for data visualization should allow the researchers to create accessible data visualization interfaces that can be exported and shared with others on the web. Herein, novel software developed for generating DNA data visualization interfaces is described. The software converts DNA data sets into images that are further processed as multi-scale images to be accessed through a web-based interface that supports zooming, panning and sequence fragment selection. Nucleotide composition frequencies and GC skew of a selected sequence segment can be obtained through the interface. The software was used to generate DNA data visualization of human and bacterial chromosomes. Examples of visually detectable features such as short and long direct repeats, long terminal repeats, mobile genetic elements, heterochromatic segments in microbial and human chromosomes, are presented. The software and its source code are available for download and further development. The visualization interfaces generated with the software allow for the immediate identification and observation of several types of sequence patterns in genomes of various sizes and origins. The visualization interfaces generated with the software are readily accessible through a web browser. This software is a useful research and teaching tool for genetics and structural genomics.

  15. Non-invasive prenatal testing using massively parallel sequencing of maternal plasma DNA: from molecular karyotyping to fetal whole-genome sequencing.

    Science.gov (United States)

    Lo, Y M Dennis

    2013-12-01

    The discovery of cell-free fetal DNA in maternal plasma in 1997 has stimulated a rapid development of non-invasive prenatal testing. The recent advent of massively parallel sequencing has allowed the analysis of circulating cell-free fetal DNA to be performed with unprecedented sensitivity and precision. Fetal trisomies 21, 18 and 13 are now robustly detectable in maternal plasma and such analyses have been available clinically since 2011. Fetal genome-wide molecular karyotyping and whole-genome sequencing have now been demonstrated in a number of proof-of-concept studies. Genome-wide and targeted sequencing of maternal plasma has been shown to allow the non-invasive prenatal testing of β-thalassaemia and can potentially be generalized to other monogenic diseases. It is thus expected that plasma DNA-based non-invasive prenatal testing will play an increasingly important role in future obstetric care. It is thus timely and important that the ethical, social and legal issues of non-invasive prenatal testing be discussed actively by all parties involved in prenatal care.

  16. A New Revised DNA Cramp Tool Based Approach of Chopping DNA Repetitive and Non-Repetitive Genome Sequences

    Directory of Open Access Journals (Sweden)

    V.Hari Prasad

    2012-11-01

    Full Text Available In vogue tremendous amount of data generated day by day by the living organism of genetic sequences and its accumulation in database, their size is growing in an exponential manner. Due to excessive storage of DNA sequences in public databases like NCBI, EMBL and DDBJ archival maintenance is tedious task. Transmission of information from one place to another place in network management systems is also a critical task. So To improve the efficiency and to reduce the overhead of the database need of compression arises in database optimization. In this connection different techniques were bloomed, but achieved results are not bountiful. Many classical algorithms are fails to compress genetic sequences due to the specificity of text encoded in dna and few of the existing techniques achieved positive results. DNA is repetitive and non repetitive in nature. Our proposed technique DNACRAMP is applicable on repetitive and non repetitive sequences of dna and it yields better compression ratio in terms of bits per bases. This is compared with existing techniques and observed that our one is the optimum technique and compression results are on par with existing techniques.

  17. 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...

  18. Deep Sequencing of Mixed Total DNA without Barcodes Allows Efficient Assembly of Highly Plastic Ascidian Mitochondrial Genomes

    Science.gov (United States)

    Rubinstein, Nimrod D.; Feldstein, Tamar; Shenkar, Noa; Botero-Castro, Fidel; Griggio, Francesca; Mastrototaro, Francesco; Delsuc, Frédéric; Douzery, Emmanuel J.P.; Gissi, Carmela; Huchon, Dorothée

    2013-01-01

    Ascidians or sea squirts form a diverse group within chordates, which includes a few thousand members of marine sessile filter-feeding animals. Their mitochondrial genomes are characterized by particularly high evolutionary rates and rampant gene rearrangements. This extreme variability complicates standard polymerase chain reaction (PCR) based techniques for molecular characterization studies, and consequently only a few complete Ascidian mitochondrial genome sequences are available. Using the standard PCR and Sanger sequencing approach, we produced the mitochondrial genome of Ascidiella aspersa only after a great effort. In contrast, we produced five additional mitogenomes (Botrylloides aff. leachii, Halocynthia spinosa, Polycarpa mytiligera, Pyura gangelion, and Rhodosoma turcicum) with a novel strategy, consisting in sequencing the pooled total DNA samples of these five species using one Illumina HiSeq 2000 flow cell lane. Each mitogenome was efficiently assembled in a single contig using de novo transcriptome assembly, as de novo genome assembly generally performed poorly for this task. Each of the new six mitogenomes presents a different and novel gene order, showing that no syntenic block has been conserved at the ordinal level (in Stolidobranchia and in Phlebobranchia). Phylogenetic analyses support the paraphyly of both Ascidiacea and Phlebobranchia, with Thaliacea nested inside Phlebobranchia, although the deepest nodes of the Phlebobranchia–Thaliacea clade are not well resolved. The strategy described here thus provides a cost-effective approach to obtain complete mitogenomes characterized by a highly plastic gene order and a fast nucleotide/amino acid substitution rate. PMID:23709623

  19. Deep sequencing of mixed total DNA without barcodes allows efficient assembly of highly plastic ascidian mitochondrial genomes.

    Science.gov (United States)

    Rubinstein, Nimrod D; Feldstein, Tamar; Shenkar, Noa; Botero-Castro, Fidel; Griggio, Francesca; Mastrototaro, Francesco; Delsuc, Frédéric; Douzery, Emmanuel J P; Gissi, Carmela; Huchon, Dorothée

    2013-01-01

    Ascidians or sea squirts form a diverse group within chordates, which includes a few thousand members of marine sessile filter-feeding animals. Their mitochondrial genomes are characterized by particularly high evolutionary rates and rampant gene rearrangements. This extreme variability complicates standard polymerase chain reaction (PCR) based techniques for molecular characterization studies, and consequently only a few complete Ascidian mitochondrial genome sequences are available. Using the standard PCR and Sanger sequencing approach, we produced the mitochondrial genome of Ascidiella aspersa only after a great effort. In contrast, we produced five additional mitogenomes (Botrylloides aff. leachii, Halocynthia spinosa, Polycarpa mytiligera, Pyura gangelion, and Rhodosoma turcicum) with a novel strategy, consisting in sequencing the pooled total DNA samples of these five species using one Illumina HiSeq 2000 flow cell lane. Each mitogenome was efficiently assembled in a single contig using de novo transcriptome assembly, as de novo genome assembly generally performed poorly for this task. Each of the new six mitogenomes presents a different and novel gene order, showing that no syntenic block has been conserved at the ordinal level (in Stolidobranchia and in Phlebobranchia). Phylogenetic analyses support the paraphyly of both Ascidiacea and Phlebobranchia, with Thaliacea nested inside Phlebobranchia, although the deepest nodes of the Phlebobranchia-Thaliacea clade are not well resolved. The strategy described here thus provides a cost-effective approach to obtain complete mitogenomes characterized by a highly plastic gene order and a fast nucleotide/amino acid substitution rate.

  20. Validated primer set that prevents nuclear DNA sequences of mitochondrial origin co-amplification: a revision based on the New Human Genome Reference Sequence (GRCh37).

    Science.gov (United States)

    Ramos, Amanda; Santos, Cristina; Barbena, Elena; Mateiu, Ligia; Alvarez, Luis; Nogués, Ramon; Aluja, Maria Pilar

    2011-03-01

    A new human genome reference sequence--GRCh37--was recently generated and made available by the Genome Reference Consortium. Since the prior disposable human reference sequence--hg18--was previously used for the mitochondrial DNA primer BLAST validation, a revision of those previously published primer pairs is required. Thus, the aim of this Short Communication is to perform an in silico BLAST test of the published disposable nine primer pairs using the new human reference sequence and to report the pertinent modifications. The new analysis showed that one of the tested primer pairs requires a revision. Therefore, a new validated primer pair, which specifically amplifies the mitochondrial region located between positions 6520 and 9184, is presented.

  1. DNA sequence comparative analysis of the 3pter-p26 region of human genome

    Institute of Scientific and Technical Information of China (English)

    LUO; Chunqing; LI; Yan; ZHANG; Xiaowei; ZHANG; Yilin; ZHAN

    2005-01-01

    Most proterminal regions of human chromosomes are GC-rich and gene-rich. Chromosome 3p is an exception. Its proterminal region is GC-poor, and likely to lose heterozygosity, thus causing a number of fatal diseases. Except one gap left in the telomeric position, the proterminal region of human chromosome 3p has been completely sequenced. The detailed sequence analysis showed: (i) the GC content of this region was 38.5%, being the lowest among all the human proterminal regions; (ii) this region contained 20 known genes and 22 predicted genes, with an average gene size of 97.5 kb. The previously mapped gene Cntn3 was not found in this region, but instead located in the 74 Mb position of human chromosome 3p; (iii) the interspersed repeats of this region were more active than the average level of the whole human genome, especially (TA)n, the content of which was twice the genome average; (iv) this region had a conserved synteny extending from 104.1 Mb to 112.4 Mb on the mouse chromosome 6, which was 8% larger in size, not in accordance with the whole genome comparison, probably because the 3pter-p26 region was more likely to lose neocleitides and its mouse synteny had more active interspersed repeats.

  2. The Complete DNA Sequence of the Mitochondrial Genome of a ``living Fossil,'' the Coelacanth (Latimeria Chalumnae)

    Science.gov (United States)

    Zardoya, R.; Meyer, A.

    1997-01-01

    The complete nucleotide sequence of the 16,407-bp mitochondrial genome of the coelacanth (Latimeria chalumnae) was determined. The coelacanth mitochondrial genome order is identical to the consensus vertebrate gene order which is also found in all ray-finned fishes, the lungfish, and most tetrapods. Base composition and codon usage also conform to typical vertebrate patterns. The entire mitochondrial genome was PCR-amplified with 24 sets of primers that are expected to amplify homologous regions in other related vertebrate species. Analyses of the control region of the coelacanth mitochondrial genome revealed the existence of four 22-bp tandem repeats close to its 3' end. The phylogenetic analyses of a large data set combining genes coding for rRNAs, tRNAs, and proteins (16,140 characters) confirmed the phylogenetic position of the coelacanth as a lobe-finned fish; it is more closely related to tetrapods than to ray-finned fishes. However, different phylogenetic methods applied to this largest available molecular data set were unable to resolve unambiguously the relationship of the coelacanth to the two other groups of extant lobe-finned fishes, the lungfishes and the tetrapods. Maximum parsimony favored a lungfish/coelacanth or a lungfish/tetrapod sistergroup relationship depending on which transversion:transition weighting is assumed. Neighbor-joining and maximum likelihood supported a lungfish/tetrapod sistergroup relationship. PMID:9215903

  3. The complete DNA sequence of the mitochondrial genome of a "living fossil," the coelacanth (Latimeria chalumnae).

    Science.gov (United States)

    Zardoya, R; Meyer, A

    1997-07-01

    The complete nucleotide sequence of the 16,407-bp mitochondrial genome of the coelacanth (Latimeria chalumnae) was determined. The coelacanth mitochondrial genome order is identical to the consensus vertebrate gene order which is also found in all ray-finned fishes, the lungfish, and most tetrapods. Base composition and codon usage also conform to typical vertebrate patterns. The entire mitochondrial genome was PCR-amplified with 24 sets of primers that are expected to amplify homologous regions in other related vertebrate species. Analyses of the control region of the coelacanth mitochondrial genome revealed the existence of four 22-bp tandem repeats close to its 3' end. The phylogenetic analyses of a large data set combining genes coding for rRNAs, tRNAs, and proteins (16,140 characters) confirmed the phylogenetic position of the coelacanth as a lobe-finned fish; it is more closely related to tetrapods than to ray-finned fishes. However, different phylogenetic methods applied to this largest available molecular data set were unable to resolve unambiguously the relationship of the coelacanth to the two other groups of extant lobe-finned fishes, the lungfishes and the tetrapods. Maximum parsimony favored a lungfish/coelacanth or a lungfish/tetrapod sistergroup relationship depending on which transversion:transition weighting is assumed. Neighbor-joining and maximum likelihood supported a lungfish/tetrapod sistergroup relationship.

  4. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

    Science.gov (United States)

    Sun, Kun; Jiang, Peiyong; Chan, K. C. Allen; Wong, John; Cheng, Yvonne K. Y.; Liang, Raymond H. S.; Chan, Wai-kong; Ma, Edmond S. K.; Chan, Stephen L.; Cheng, Suk Hang; Chan, Rebecca W. Y.; Tong, Yu K.; Ng, Simon S. M.; Wong, Raymond S. M.; Hui, David S. C.; Leung, Tse Ngong; Leung, Tak Y.; Lai, Paul B. S.; Chiu, Rossa W. K.; Lo, Yuk Ming Dennis

    2015-01-01

    Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma. PMID:26392541

  5. Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing

    Science.gov (United States)

    Chan, K. C. Allen; Jiang, Peiyong; Chan, Carol W. M.; Sun, Kun; Wong, John; Hui, Edwin P.; Chan, Stephen L.; Chan, Wing Cheong; Hui, David S. C.; Ng, Simon S. M.; Chan, Henry L. Y.; Wong, Cesar S. C.; Ma, Brigette B. Y.; Chan, Anthony T. C.; Lai, Paul B. S.; Sun, Hao; Chiu, Rossa W. K.; Lo, Y. M. Dennis

    2013-01-01

    We explored the detection of genome-wide hypomethylation in plasma using shotgun massively parallel bisulfite sequencing as a marker for cancer. Tumor-associated copy number aberrations (CNAs) could also be observed from the bisulfite DNA sequencing data. Hypomethylation and CNAs were detected in the plasma DNA of patients with hepatocellular carcinoma, breast cancer, lung cancer, nasopharyngeal cancer, smooth muscle sarcoma, and neuroendocrine tumor. For the detection of nonmetastatic cancer cases, plasma hypomethylation gave a sensitivity and specificity of 74% and 94%, respectively, when a mean of 93 million reads per case were obtained. Reducing the sequencing depth to 10 million reads per case was found to have no adverse effect on the sensitivity and specificity for cancer detection, giving respective figures of 68% and 94%. This characteristic thus indicates that analysis of plasma hypomethylation by this sequencing-based method may be a relatively cost-effective approach for cancer detection. We also demonstrated that plasma hypomethylation had utility for monitoring hepatocellular carcinoma patients following tumor resection and for detecting residual disease. Plasma hypomethylation can be combined with plasma CNA analysis for further enhancement of the detection sensitivity or specificity using different diagnostic algorithms. Using the detection of at least one type of aberration to define an abnormality, a sensitivity of 87% could be achieved with a specificity of 88%. These developments have thus expanded the applications of plasma DNA analysis for cancer detection and monitoring. PMID:24191000

  6. Complete Genome Sequence of Pelosinus sp. Strain UFO1 Assembled Using Single-Molecule Real-Time DNA Sequencing Technology

    Energy Technology Data Exchange (ETDEWEB)

    Steven D. Brown; Sagar M. Utturkar; Timothy S. Magnuson; Allison E. Ray; Farris L. Poole; W. Andrew Lancaster; Michael P. Thorgersen; Michael W. W. Adams; Dwayne A. Elias

    2014-09-01

    Pelosinus fermentans strain R7 was isolated from Russian kaolin clays as the type strain and it can reduce Fe(III) during fermentative growth (1). Draft genome sequences for P. fermentans R7 and four strains from Hanford, Washington, USA, have been published (2–4). The P. fermentans 16S rRNA sequence dominated the lactate-based enrichment cultures from three geochemically contrasting soils from the Melton Branch Watershed, Oak Ridge, Tennessee, USA (5) and also at another stimulated, uraniumcontaminated field site near Oak Ridge (6). For the current work, strain UFO1 was isolated from pristine sediments at a background field site in Oak Ridge and characterized as facilitating U(VI) reduction and precipitation with phosphate (7).

  7. epiG: statistical inference and profiling of DNA methylation from whole-genome bisulfite sequencing data.

    Science.gov (United States)

    Vincent, Martin; Mundbjerg, Kamilla; Skou Pedersen, Jakob; Liang, Gangning; Jones, Peter A; Ørntoft, Torben Falck; Dalsgaard Sørensen, Karina; Wiuf, Carsten

    2017-02-21

    The study of epigenetic heterogeneity at the level of individual cells and in whole populations is the key to understanding cellular differentiation, organismal development, and the evolution of cancer. We develop a statistical method, epiG, to infer and differentiate between different epi-allelic haplotypes, annotated with CpG methylation status and DNA polymorphisms, from whole-genome bisulfite sequencing data, and nucleosome occupancy from NOMe-seq data. We demonstrate the capabilities of the method by inferring allele-specific methylation and nucleosome occupancy in cell lines, and colon and tumor samples, and by benchmarking the method against independent experimental data.

  8. Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip

    Directory of Open Access Journals (Sweden)

    Carr Steven M

    2007-09-01

    Full Text Available Abstract Background Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific biodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. Results In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands ≥ 12b that are conserved between primates and fish. Conclusion Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by

  9. Genbit Compress Tool(GBC): A Java-Based Tool to Compress DNA Sequences and Compute Compression Ratio(bits/base) of Genomes

    CERN Document Server

    Rajeswari, P Raja; Kumar, V K; 10.5121/ijcsit.2010.2313

    2010-01-01

    We present a Compression Tool, "GenBit Compress", for genetic sequences based on our new proposed "GenBit Compress Algorithm". Our Tool achieves the best compression ratios for Entire Genome (DNA sequences) . Significantly better compression results show that GenBit compress algorithm is the best among the remaining Genome compression algorithms for non-repetitive DNA sequences in Genomes. The standard Compression algorithms such as gzip or compress cannot compress DNA sequences but only expand them in size. In this paper we consider the problem of DNA compression. It is well known that one of the main features of DNA Sequences is that they contain substrings which are duplicated except for a few random Mutations. For this reason most DNA compressors work by searching and encoding approximate repeats. We depart from this strategy by searching and encoding only exact repeats. our proposed algorithm achieves the best compression ratio for DNA sequences for larger genome. As long as 8 lakh characters can be give...

  10. K-mer Content, Correlation, and Position Analysis of Genome DNA Sequences for the Identification of Function and Evolutionary Features.

    Science.gov (United States)

    Sievers, Aaron; Bosiek, Katharina; Bisch, Marc; Dreessen, Chris; Riedel, Jascha; Froß, Patrick; Hausmann, Michael; Hildenbrand, Georg

    2017-04-19

    In genome analysis, k-mer-based comparison methods have become standard tools. However, even though they are able to deliver reliable results, other algorithms seem to work better in some cases. To improve k-mer-based DNA sequence analysis and comparison, we successfully checked whether adding positional resolution is beneficial for finding and/or comparing interesting organizational structures. A simple but efficient algorithm for extracting and saving local k-mer spectra (frequency distribution of k-mers) was developed and used. The results were analyzed by including positional information based on visualizations as genomic maps and by applying basic vector correlation methods. This analysis was concentrated on small word lengths (1 ≤ k ≤ 4) on relatively small viral genomes of Papillomaviridae and Herpesviridae, while also checking its usability for larger sequences, namely human chromosome 2 and the homologous chromosomes (2A, 2B) of a chimpanzee. Using this alignment-free analysis, several regions with specific characteristics in Papillomaviridae and Herpesviridae formerly identified by independent, mostly alignment-based methods, were confirmed. Correlations between the k-mer content and several genes in these genomes have been found, showing similarities between classified and unclassified viruses, which may be potentially useful for further taxonomic research. Furthermore, unknown k-mer correlations in the genomes of Human Herpesviruses (HHVs), which are probably of major biological function, are found and described. Using the chromosomes of a chimpanzee and human that are currently known, identities between the species on every analyzed chromosome were reproduced. This demonstrates the feasibility of our approach for large data sets of complex genomes. Based on these results, we suggest k-mer analysis with positional resolution as a method for closing a gap between the effectiveness of alignment-based methods (like NCBI BLAST) and the high pace of

  11. Whole Genome DNA Sequence Analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

    Science.gov (United States)

    Wilson, Mark R; Brown, Eric; Keys, Chris; Strain, Errol; Luo, Yan; Muruvanda, Tim; Grim, Christopher; Jean-Gilles Beaubrun, Junia; Jarvis, Karen; Ewing, Laura; Gopinath, Gopal; Hanes, Darcy; Allard, Marc W; Musser, Steven

    2016-01-01

    Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS) to Salmonella subspecies enterica serotype Tennessee (S. Tennessee) to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana), which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP) analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs), suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future

  12. Whole Genome DNA Sequence Analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

    Directory of Open Access Journals (Sweden)

    Mark R Wilson

    Full Text Available Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS to Salmonella subspecies enterica serotype Tennessee (S. Tennessee to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana, which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs, suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts

  13. Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

    NARCIS (Netherlands)

    Zheng, S.J.; Henken, G.; Sofiari, E.; Jacobsen, E.; Krens, F.A.

    2001-01-01

    Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a

  14. Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines

    Directory of Open Access Journals (Sweden)

    Cornish Katrina

    2009-11-01

    Full Text Available Abstract Background Parthenium argentatum (guayule is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of P. argentatum was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, Lactuca sativa, Guitozia abyssinica and Helianthus annuus revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of Parthenium species and lines. Results The complete plastid genome of P. argentatum is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in L. sativa, G. abyssinica and H. annuus, we demonstrate that the P. argentatum chloroplast genome sequence is most closely related to that of H. annuus. Similar to chloroplast genomes in G. abyssinica, L. sativa and H. annuus, the plastid genome of P. argentatum has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the matK and psbA-trnH spacer chloroplast DNA barcodes, three of the four Parthenium species tested, P. tomentosum, P. hysterophorus and P. schottii, can be differentiated from P. argentatum. In addition, we identified lines within P. argentatum. Conclusion The genome sequence of the P. argentatum chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate transformation efficiency by using the precise sequence of endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially significant lines of P. argentatum that are important for producing latex.

  15. An optimized and low-cost FPGA-based DNA sequence alignment--a step towards personal genomics.

    Science.gov (United States)

    Shah, Hurmat Ali; Hasan, Laiq; Ahmad, Nasir

    2013-01-01

    DNA sequence alignment is a cardinal process in computational biology but also is much expensive computationally when performing through traditional computational platforms like CPU. Of many off the shelf platforms explored for speeding up the computation process, FPGA stands as the best candidate due to its performance per dollar spent and performance per watt. These two advantages make FPGA as the most appropriate choice for realizing the aim of personal genomics. The previous implementation of DNA sequence alignment did not take into consideration the price of the device on which optimization was performed. This paper presents optimization over previous FPGA implementation that increases the overall speed-up achieved as well as the price incurred by the platform that was optimized. The optimizations are (1) The array of processing elements is made to run on change in input value and not on clock, so eliminating the need for tight clock synchronization, (2) the implementation is unrestrained by the size of the sequences to be aligned, (3) the waiting time required for the sequences to load to FPGA is reduced to the minimum possible and (4) an efficient method is devised to store the output matrix that make possible to save the diagonal elements to be used in next pass, in parallel with the computation of output matrix. Implemented on Spartan3 FPGA, this implementation achieved 20 times performance improvement in terms of CUPS over GPP implementation.

  16. The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny.

    Science.gov (United States)

    Williams, S T; Foster, P G; Littlewood, D T J

    2014-01-01

    A need to increase sampling of mitochondrial genomes for Vetigastropoda has been identified as an important step towards resolving relationships within the Gastropoda. We used shotgun sequencing of genomic DNA, using an Illumina MiSeq, to obtain the first mitochondrial genome for the vetigastropod family Turbinidae, doubling the number of genomes for the species-rich superfamily Trochoidea. This method avoids the necessity of finding suitable primers for long PCRs or primer-walking amplicons, resulting in a timely and cost-effective method for obtaining whole mitochondrial genomes from ethanol-preserved tissue samples. Bayesian analysis of amino acid variation for all available gastropod genomes including the new turbinid mtgenome produced a well resolved tree with high nodal support for most nodes. Major clades within Gastropoda were recovered with strong support, with the exception of Littorinimorpha, which was polyphyletic. We confirm here that mitogenomics is a useful tool for molluscan phylogenetics, especially when using powerful new models of amino acid evolution, but recognise that increased taxon sampling is still required to resolve existing differences between nuclear and mitochondrial gene trees.

  17. Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

    Directory of Open Access Journals (Sweden)

    Rodrigues NB

    2002-01-01

    Full Text Available In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3% sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds. Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8% contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds. The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds. From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

  18. Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries.

    Science.gov (United States)

    Rodrigues, N B; Loverde, P T; Romanha, A J; Oliveira, G

    2002-01-01

    In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

  19. Yeast genome sequencing:

    DEFF Research Database (Denmark)

    Piskur, Jure; Langkjær, Rikke Breinhold

    2004-01-01

    For decades, unicellular yeasts have been general models to help understand the eukaryotic cell and also our own biology. Recently, over a dozen yeast genomes have been sequenced, providing the basis to resolve several complex biological questions. Analysis of the novel sequence data has shown...... of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because...... they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...

  20. Organellar genome, nuclear ribosomal DNA repeat unit, and microsatellites isolated from a small-scale of 454 GS FLX sequencing on two mosses.

    Science.gov (United States)

    Liu, Yang; Forrest, Laura L; Bainard, Jillian D; Budke, Jessica M; Goffinet, Bernard

    2013-03-01

    Recent innovations in high-throughput DNA sequencing methodology (next generation sequencing technologies [NGS]) allow for the generation of large amounts of high quality data that may be particularly critical for resolving ambiguous relationships such as those resulting from rapid radiations. Application of NGS technology to bryology is limited to assembling entire nuclear or organellar genomes of selected exemplars of major lineages (e.g., classes). Here we outline how organellar genomes and the entire nuclear ribosomal DNA repeat can be obtained from minimal amounts of moss tissue via small-scale 454 GS FLX sequencing. We sampled two Funariaceae species, Funaria hygrometrica and Entosthodon obtusus, and assembled nearly complete organellar genomes and the whole nuclear ribosomal DNA repeat unit (18S-ITS1-5.8S-ITS2-26S-IGS1-5S-IGS2) for both taxa. Sequence data from these species were compared to sequences from another Funariaceae species, Physcomitrella patens, revealing low overall degrees of divergence of the organellar genomes and nrDNA genes with substitutions spread rather evenly across their length, and high divergence within the external spacers of the nrDNA repeat. Furthermore, we detected numerous microsatellites among the 454 assemblies. This study demonstrates that NGS methodology can be applied to mosses to target large genomic regions and identify microsatellites.

  1. Next-Gen phylogeography of rainforest trees: exploring landscape-level cpDNA variation from whole-genome sequencing.

    Science.gov (United States)

    van der Merwe, M; McPherson, H; Siow, J; Rossetto, M

    2014-01-01

    Standardized phylogeographic studies across codistributed taxa can identify important refugia and biogeographic barriers, and potentially uncover how changes in adaptive constraints through space and time impact on the distribution of genetic diversity. The combination of next-generation sequencing and methodologies that enable uncomplicated analysis of the full chloroplast genome may provide an invaluable resource for such studies. Here, we assess the potential of a shotgun-based method across twelve nonmodel rainforest trees sampled from two evolutionary distinct regions. Whole genomic shotgun sequencing libraries consisting of pooled individuals were used to assemble species-specific chloroplast references (in silicio). For each species, the pooled libraries allowed for the detection of variation within and between data sets (each representing a geographic region). The potential use of nuclear rDNA as an additional marker from the NGS libraries was investigated by mapping reads against available references. We successfully obtained phylogeographically informative sequence data from a range of previously unstudied rainforest trees. Greater levels of diversity were found in northern refugial rainforests than in southern expansion areas. The genetic signatures of varying evolutionary histories were detected, and interesting associative patterns between functional characteristics and genetic diversity were identified. This approach can suit a wide range of landscape-level studies. As the key laboratory-based steps do not require prior species-specific knowledge and can be easily outsourced, the techniques described here are even suitable for researchers without access to wet-laboratory facilities, making evolutionary ecology questions increasingly accessible to the research community.

  2. Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution

    Directory of Open Access Journals (Sweden)

    Kishima Yuji

    2004-10-01

    Full Text Available Abstract Background Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV-like sequences (ERTBVs have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response. Results We have collected ERTBVs from the rice genomes. They contain rearranged structures and no intact ORFs. The identified ERTBV segments were shown to be phylogenetically divided into three clusters. For each phylogenetic cluster, we were able to make a consensus alignment for a circular virus-like structure carrying two complete ORFs. Comparisons of DNA and amino acid sequences suggested the closely relationship between ERTBV and RTBV. The Oryza AA-genome species vary in the ERTBV copy number. The species carrying low-copy-number of ERTBV segments have been reported to be extremely susceptible to RTBV. The DNA methylation state of the ERTBV sequences was correlated with their copy number in the genome. Conclusions These ERTBV segments are unlikely to have functional potential as a virus. However, these sequences facilitate to establish putative virus that provided information underlying virus integration and evolutionary relationship with existing virus. Comparison of ERTBV among the Oryza AA-genome species allowed us to speculate a possible role of endogenous virus segments against its related disease.

  3. Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies.

    Science.gov (United States)

    Michael, Todd P; Bryant, Douglas; Gutierrez, Ryan; Borisjuk, Nikolai; Chu, Philomena; Zhang, Hanzhong; Xia, Jing; Zhou, Junfei; Peng, Hai; El Baidouri, Moaine; Ten Hallers, Boudewijn; Hastie, Alex R; Liang, Tiffany; Acosta, Kenneth; Gilbert, Sarah; McEntee, Connor; Jackson, Scott A; Mockler, Todd C; Zhang, Weixiong; Lam, Eric

    2017-02-01

    Spirodela polyrhiza is a fast-growing aquatic monocot with highly reduced morphology, genome size and number of protein-coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158-Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome-wide physical maps combined with high-coverage short-read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela-specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non-essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large-scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  4. DNA sequences encoding erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, F.K.

    1987-10-27

    A purified and isolated DNA sequence is described consisting essentially of a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of that of erythropoietin to allow possession of the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells, and to increase hemoglobin synthesis or iron uptake.

  5. cDNA, genomic sequence cloning and overexpression of ribosomal protein S25 gene (RPS25) from the Giant Panda.

    Science.gov (United States)

    Hao, Yan-Zhe; Hou, Wan-Ru; Hou, Yi-Ling; Du, Yu-Jie; Zhang, Tian; Peng, Zheng-Song

    2009-11-01

    RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a "living fossil", are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first

  6. Genomic sequencing of Pleistocene cave bears

    Energy Technology Data Exchange (ETDEWEB)

    Noonan, James P.; Hofreiter, Michael; Smith, Doug; Priest, JamesR.; Rohland, Nadin; Rabeder, Gernot; Krause, Johannes; Detter, J. Chris; Paabo, Svante; Rubin, Edward M.

    2005-04-01

    Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of {approx}1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome, the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.

  7. [Genomic DNA sequences and functional expression, purification of BmalphaTX14 neurotoxin from scorpion Buthus martensii Karsch].

    Science.gov (United States)

    Lü, Meng; Wang, Kun; Cao, Zhi-Jian; Jiang, Da-He; Mao, Xin; Li, Wen-Xin

    2005-11-01

    Based on the full-length cDNA of BmalphaTX14 from Chinese scorpion Buthus martensii Karsch (BmK), gene of the mature peptide of BmalphaTX14 was cloned into the yeast expression vector pPIC9K. After transforming, screening and inducing, tricine-SDS-PAGE and Western blot proved that rBmalphaTX14 protein was expressed in the medium for up to 84 hours, getting nearly 120 mg/L. Recombinant BmalphaTX14 was purified rapidly and efficiently through Ni-NTA-agarose, polyethylene glycol precipitation and gel filtration chromatography. The purified rBmalphaTX14 proved to have the anti-insect activity by toxicity assay. Meanwhile, genomic gene of BmalphaTX14 was cloned and sequenced by PCR method, sequence analysis of this gene showed that BmalphaTX14 had an intron of 408 base pairs located at the signal peptide encoding region, which was similar with the characteristic of other alpha-type sodium ion-channel toxin. Considering both the genomic organization and the peptide function, BmaTX14 proved to be a membership belonging to alpha-type sodium ion-channel toxin.

  8. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  9. Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing

    Directory of Open Access Journals (Sweden)

    ElSharawy Abdou

    2012-09-01

    Full Text Available Abstract Background Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA. We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS workflow. Results The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. Conclusion Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.

  10. Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

    Directory of Open Access Journals (Sweden)

    Yukio Kurihara

    2014-12-01

    Full Text Available Several transcription factors (TFs coordinate to regulate expression of specific genes at the transcriptional level. In Arabidopsis thaliana it is estimated that approximately 10% of all genes encode TFs or TF-like proteins. It is important to identify target genes that are directly regulated by TFs in order to understand the complete picture of a plant’s transcriptome profile. Here, we investigate the role of the LONG HYPOCOTYL5 (HY5 transcription factor that acts as a regulator of photomorphogenesis. We used an in vitro genomic DNA binding assay coupled with immunoprecipitation and next-generation sequencing (gDB-seq instead of the in vivo chromatin immunoprecipitation (ChIP-based methods. The results demonstrate that the HY5-binding motif predicted here was similar to the motif reported previously and that in vitro HY5-binding loci largely overlapped with the HY5-targeted candidate genes identified in previous ChIP-chip analysis. By combining these results with microarray analysis, we identified hundreds of HY5-binding genes that were differentially expressed in hy5. We also observed delayed induction of some transcripts of HY5-binding genes in hy5 mutants in response to blue-light exposure after dark treatment. Thus, an in vitro gDNA-binding assay coupled with sequencing is a convenient and powerful method to bridge the gap between identifying TF binding potential and establishing function.

  11. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    John A Capra

    2010-07-01

    Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  12. Characterization of the genome of molluscum contagiosum virus type 1 between the genome coordinates 0.045 and 0.075 by DNA nucleotide sequence analysis of a 5.6-kb HindIII/MluI DNA fragment.

    Science.gov (United States)

    Hadasch, R P; Bugert, J J; Janssen, W; Darai, G

    1993-01-01

    The complete DNA nucleotide sequence of a HindIII/MluI genomic DNA fragment (0.045-0.075 viral map units) from molluscum contagiosum virus type 1 (MCV-1) was determined. The HindIII/MluI DNA fragment comprises 5,646 bp with a base composition of 64.4% G + C and 35.6% A + T. The DNA sequence contains many perfect direct repeats. A cluster of three repetitive DNA elements R1, R2 and R3, with a complex structural arrangement was detected between nucleotide positions 1802 and 2107. The unit length (box) of the repetitive DNA sequences was found to be 6 bp (15 boxes) and 9 bp (24 boxes) for R1 and R2, respectively. The repetitive DNA element R3 is organized in fifteen boxes (15 bp) in which a unit length of R1 is combined with a unit length of R2. The arrangement of the repetition R3 within the DNA sequences of this particular region of the MCV-1 genome was found to be (5 x R3) + (2 x R2) + (1 x R3) + (6 x R2) + (1 x R3) + (1 x R2) + (8 x R3). Twenty-three open reading frames (ORFs) of 60-1,175 amino acid (AA) residues were detected. The largest ORF (number 17) comprises 1,175 AA with a predicted molecular weight of 126 kD. This ORF harbors a promoter signal which is located 21 nucleotides upstream from the start codon and is very similar to the early promoter signals known for vaccinia virus. This putative protein contains glutamine-enriched regions between AA residues 427 and 682 which show homologies to the corresponding glutamine-enriched regions of a variety of cellular genes like human transcriptional initiation factor (TFIID: TATA box factor).

  13. Genome Sequence of Mycobacteriophage Momo.

    Science.gov (United States)

    Pope, Welkin H; Bina, Elizabeth A; Brahme, Indraneel S; Hill, Amy B; Himmelstein, Philip H; Hunsicker, Sara M; Ish, Amanda R; Le, Tinh S; Martin, Mary M; Moscinski, Catherine N; Shetty, Sameer A; Swierzewski, Tomasz; Iyengar, Varun B; Kim, Hannah; Schafer, Claire E; Grubb, Sarah R; Warner, Marcie H; Bowman, Charles A; Russell, Daniel A; Hatfull, Graham F

    2015-06-18

    Momo is a newly discovered phage of Mycobacterium smegmatis mc(2)155. Momo has a double-stranded DNA genome 154,553 bp in length, with 233 predicted protein-encoding genes, 34 tRNA genes, and one transfer-messenger RNA (tmRNA) gene. Momo has a myoviral morphology and shares extensive nucleotide sequence similarity with subcluster C1 mycobacteriophages. Copyright © 2015 Pope et al.

  14. Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data.

    Science.gov (United States)

    Soorni, Aboozar; Haak, David; Zaitlin, David; Bombarely, Aureliano

    2017-01-07

    The development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present. We present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and then performs read correction and de novo assembly using Sprai. Organelle-PBA completes the assembly process with the additional step of scaffolding by SSPACE-LongRead. The program then detects the chloroplast inverted repeats and reassembles and re-orients the assembly based on the organelle origin of the reference. We have evaluated the performance of the software using PacBio reads from different species, read coverage, and reference genomes. Finally, we present the assembly of two novel chloroplast genomes from the species Picea glauca (Pinaceae) and Sinningia speciosa (Gesneriaceae). Organelle-PBA is an easy-to-use Perl-based software pipeline that was written specifically to assemble mitochondrial and chloroplast genomes from whole genome PacBio reads. The program is available at https://github.com/aubombarely/Organelle_PBA .

  15. New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes for radiation molecular cytogenetics

    Directory of Open Access Journals (Sweden)

    Repin Mikhail V

    2009-06-01

    Full Text Available Abstract Background The objective of this work is to obtain the correct relative DNA contents of chromosomes in the normal male and female human diploid genomes for the use at FISH analysis of radiation-induced chromosome aberrations. Results The relative DNA contents of chromosomes in the male and female human diploid genomes have been calculated from the publicly available international Human Genome Project data. New sequence-based data on the relative DNA contents of human chromosomes were compared with the data recommended by the International Atomic Energy Agency in 2001. The differences in the values of the relative DNA contents of chromosomes obtained by using different approaches for 15 human chromosomes, mainly for large chromosomes, were below 2%. For the chromosomes 13, 17, 20 and 22 the differences were above 5%. Conclusion New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes were obtained. This approach, based on the genome sequence, can be recommended for the use in radiation molecular cytogenetics.

  16. A Bayesian Approach for Analysis of Whole-Genome Bisulphite Sequencing Data Identifies Disease-Associated Changes in DNA Methylation.

    Science.gov (United States)

    Rackham, Owen J L; Langley, Sarah R; Oates, Thomas; Vradi, Eleni; Harmston, Nathan; Srivastava, Prashant K; Behmoaras, Jacques; Dellaportas, Petros; Bottolo, Leonardo; Petretto, Enrico

    2017-02-17

    DNA methylation is a key epigenetic modification involved in gene regulation whose contribution to disease susceptibility remains to be fully understood. Here, we present a novel Bayesian smoothing approach (called ABBA) to detect differentially methylated regions (DMRs) from whole-genome bisulphite sequencing (WGBS). We also show how this approach can be leveraged to identify disease-associated changes in DNA methylation, suggesting mechanisms through which these alterations might affect disease. From a data modeling perspective, ABBA has the distinctive feature of automatically adapting to different correlation structures in CpG methylation levels across the genome whilst taking into account the distance between CpG sites as a covariate. Our simulation study shows that ABBA has greater power to detect DMRs than existing methods, providing an accurate identification of DMRs in the large majority of simulated cases. To empirically demonstrate the method's efficacy in generating biological hypotheses, we performed WGBS of primary macrophages derived from an experimental rat system of glomerulonephritis and used ABBA to identify >1,000 disease-associated DMRs. Investigation of these DMRs revealed differential DNA methylation localized to a 600bp region in the promoter of the Ifitm3 gene. This was confirmed by ChIP-seq and RNA-seq analyses, showing differential transcription factor binding at the Ifitm3 promoter by JunD (an established determinant of glomerulonephritis) and a consistent change in Ifitm3 expression. Our ABBA analysis allowed us to propose a new role for Ifitm3 in the pathogenesis of glomerulonephritis via a mechanism involving promoter hypermethylation that is associated with Ifitm3 repression in the rat strain susceptible to glomerulonephritis.

  17. Microbial genomics: from sequence to function.

    OpenAIRE

    Schwartz, I

    2000-01-01

    The era of genomics (the study of genes and their function) began a scant dozen years ago with a suggestion by James Watson that the complete DNA sequence of the human genome be determined. Since that time, the human genome project has attracted a great deal of attention in the scientific world and the general media; the scope of the sequencing effort, and the extraordinary value that it will provide, has served to mask the enormous progress in sequencing other genomes. Microbial genome seque...

  18. Intermittency as a universal characteristic of the complete chromosome DNA sequences of eukaryotes: From protozoa to human genomes

    Science.gov (United States)

    Rybalko, S.; Larionov, S.; Poptsova, M.; Loskutov, A.

    2011-10-01

    Large-scale dynamical properties of complete chromosome DNA sequences of eukaryotes are considered. Using the proposed deterministic models with intermittency and symbolic dynamics we describe a wide spectrum of large-scale patterns inherent in these sequences, such as segmental duplications, tandem repeats, and other complex sequence structures. It is shown that the recently discovered gene number balance on the strands is not of a random nature, and certain subsystems of a complete chromosome DNA sequence exhibit the properties of deterministic chaos.

  19. DNA Sequencing Sensors: An Overview

    Directory of Open Access Journals (Sweden)

    Jose Antonio Garrido-Cardenas

    2017-03-01

    Full Text Available The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years.

  20. Extensive sequence-influenced DNA methylation polymorphism in the human genome

    OpenAIRE

    Hellman Asaf; Chess Andrew

    2010-01-01

    Abstract Background Epigenetic polymorphisms are a potential source of human diversity, but their frequency and relationship to genetic polymorphisms are unclear. DNA methylation, an epigenetic mark that is a covalent modification of the DNA itself, plays an important role in the regulation of gene expression. Most studies of DNA methylation in mammalian cells have focused on CpG methylation present in CpG islands (areas of concentrated CpGs often found near promoters), but there are also int...

  1. SigWin-detector: a Grid-enabled workflow for discovering enriched windows of genomic features related to DNA sequences

    Directory of Open Access Journals (Sweden)

    Wibisono Adianto

    2008-08-01

    Full Text Available Abstract Background Chromosome location is often used as a scaffold to organize genomic information in both the living cell and molecular biological research. Thus, ever-increasing amounts of data about genomic features are stored in public databases and can be readily visualized by genome browsers. To perform in silico experimentation conveniently with this genomics data, biologists need tools to process and compare datasets routinely and explore the obtained results interactively. The complexity of such experimentation requires these tools to be based on an e-Science approach, hence generic, modular, and reusable. A virtual laboratory environment with workflows, workflow management systems, and Grid computation are therefore essential. Findings Here we apply an e-Science approach to develop SigWin-detector, a workflow-based tool that can detect significantly enriched windows of (genomic features in a (DNA sequence in a fast and reproducible way. For proof-of-principle, we utilize a biological use case to detect regions of increased and decreased gene expression (RIDGEs and anti-RIDGEs in human transcriptome maps. We improved the original method for RIDGE detection by replacing the costly step of estimation by random sampling with a faster analytical formula for computing the distribution of the null hypothesis being tested and by developing a new algorithm for computing moving medians. SigWin-detector was developed using the WS-VLAM workflow management system and consists of several reusable modules that are linked together in a basic workflow. The configuration of this basic workflow can be adapted to satisfy the requirements of the specific in silico experiment. Conclusion As we show with the results from analyses in the biological use case on RIDGEs, SigWin-detector is an efficient and reusable Grid-based tool for discovering windows enriched for features of a particular type in any sequence of values. Thus, SigWin-detector provides the

  2. Malaria Genome Sequencing Project

    Science.gov (United States)

    2004-01-01

    million cases and up to 2.7 million A whole chromosome shotgun sequencing strategy was used to deaths from malaria each year. The mortality levels are...deaths from malaria each year. The mortality levels are greatest in determine the genome sequence of P. falciparum clone 3D7. This sub-Saharan Africa...aminolevulinic acid dehydratase. Cura . Genet. 40, 391-398 (2002). 15. Lasonder, E. et al Analysis of the Plasmodium falciparum proteome by high-accuracy mass

  3. Automated DNA Sequencing System

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  4. Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

    Science.gov (United States)

    Kader, Tanjina; Goode, David L; Wong, Stephen Q; Connaughton, Jacquie; Rowley, Simone M; Devereux, Lisa; Byrne, David; Fox, Stephen B; Mir Arnau, Gisela; Tothill, Richard W; Campbell, Ian G; Gorringe, Kylie L

    2016-11-15

    Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.

  5. Genetic integrity of the Dark European honey bee (Apis mellifera mellifera) from protected populations: a genome-wide assessment using SNPs and mtDNA sequence data

    DEFF Research Database (Denmark)

    Pinto, M Alice; Henriques, Dora; Chávez-Galarza, Julio

    2014-01-01

    to preserve the genetic integrity of A. m. mellifera, protected populations had a measurable component of their gene pool derived from commercial C-lineage honey bees. Here we used both sequence data from the tRNAleu-cox2 intergenic mtDNA region and a genome-wide scan, with over 1183 single nucleotide...

  6. Genetic integrity of the Dark European honey bee (Apis mellifera mellifera) from protected populations: a genome-wide assessment using SNPs and mtDNA sequence data

    DEFF Research Database (Denmark)

    Pinto, M Alice; Henriques, Dora; Chávez-Galarza, Julio

    2014-01-01

    to preserve the genetic integrity of A. m. mellifera, protected populations had a measurable component of their gene pool derived from commercial C-lineage honey bees. Here we used both sequence data from the tRNAleu-cox2 intergenic mtDNA region and a genome-wide scan, with over 1183 single nucleotide...

  7. Sequencing the maize genome.

    Science.gov (United States)

    Martienssen, Robert A; Rabinowicz, Pablo D; O'Shaughnessy, Andrew; McCombie, W Richard

    2004-04-01

    Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.

  8. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR ... RNA species and approximately 80 structurally distinct ... a reverse transcription kit with Oligo dT as the primers according to ... 2). On the basis of the high identity, we concluded that the cDNA isolated is ...

  9. DNA fingerprinting of Mycobacterium tuberculosis: from phage typing to whole-genome sequencing.

    NARCIS (Netherlands)

    Schurch, A.C.; Soolingen, D. van

    2012-01-01

    Current typing methods for Mycobacterium tuberculosis complex evolved from simple phenotypic approaches like phage typing and drug susceptibility profiling to DNA-based strain typing methods, such as IS6110-restriction fragment length polymorphisms (RFLP) and variable number of tandem repeats (VNTR)

  10. Repetitive DNA in the pea (Pisum sativum L. genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula

    Directory of Open Access Journals (Sweden)

    Navrátilová Alice

    2007-11-01

    Full Text Available Abstract Background Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum. Results Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. Conclusion We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35–48% of the genome. These data

  11. Isolation of genomic DNA from mammalian cells.

    Science.gov (United States)

    Koh, Cheryl M

    2013-01-01

    The isolation of genomic DNA from mammalian cells is a routine molecular biology laboratory technique with numerous downstream applications. The isolated DNA can be used as a template for PCR, cloning, and genotyping and to generate genomic DNA libraries. It can also be used for sequencing to detect mutations and other alterations, and for DNA methylation analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Evolution of DNA sequencing

    National Research Council Canada - National Science Library

    Tipu, Hamid Nawaz; Shabbir, Ambreen

    2015-01-01

    Sanger and coworkers introduced DNA sequencing in 1970s for the first time. It principally relied on termination of growing nucleotide chain when a dideoxythymidine triphosphate (ddTTP) was inserted...

  13. Gomphid DNA sequence data

    Data.gov (United States)

    U.S. Environmental Protection Agency — DNA sequence data for several genetic loci. This dataset is not publicly accessible because: It's already publicly available on GenBank. It can be accessed through...

  14. Comparison of the tyrosine aminotransferase cDNA and genomic DNA sequences of normal mink and mink affected with tyrosinemia type II.

    Science.gov (United States)

    Leib, S R; McGuire, T C; Prieur, D J

    2005-01-01

    Type II tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of tyrosine aminotransferase activity. Mutations occur in the human tyrosine aminotransferase gene, resulting in high levels of tyrosine and disease. Type II tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the tyrosine aminotransferase gene. Therefore, the transcribed cDNA and the genomic tyrosine aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human tyrosine aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of tyrosinemia in mink.

  15. DNA sequence recognition by hybridization to short oligomers : experimental determination of accuracy of the method in a genome-scale search.

    Energy Technology Data Exchange (ETDEWEB)

    Milosavljevic, A.; Savkovic, S.; Crkvenjakov, R.; Salbego, D.; Serrato, H.; Kreuzer, H.; Gemmell, A.; Batus, S.; Grujic, D.; Carnahan, S.; Tepavcevic, J.; Center for Mechanistic Biology and Biotechnology

    1996-01-01

    Recently developed hybridization technology enables economical large-scale detection of short oligomers within DNA fragments. The newly developed recognition method enables comparison of lists of oligomers detected within DNA fragments against known DNA sequences. We here describe an experiment involving a set of 4513 distinct genomic E. coli clones of average length 2kb, each hybridized with 636 randomly selected short oligomer probes. High hybridization signal with a particular probe was used as an indication of the presence of a complementary oligomer in the particular clone. For each clone, a list of oligomers with highest hybridization signals was compiled. The database consisting of 4513 oligomer lists was then searched using known E. coli sequences as queries in an attempt to identify the clones that match the query sequence. Out of a total of 11 clones that were recognized at highest significance level by our method, 8 were single-pass sequenced from both ends. The single-pass sequenced ends were then compared against the query sequences. The sequence comparisons confirmed 7 out of the total of 8 examined recognitions. This experiment represents the first successful example of genome-scale sequence recognition based on hybridization data.

  16. Maize genome sequencing by methylation filtration.

    Science.gov (United States)

    Palmer, Lance E; Rabinowicz, Pablo D; O'Shaughnessy, Andrew L; Balija, Vivekanand S; Nascimento, Lidia U; Dike, Sujit; de la Bastide, Melissa; Martienssen, Robert A; McCombie, W Richard

    2003-12-19

    Gene enrichment strategies offer an alternative to sequencing large and repetitive genomes such as that of maize. We report the generation and analysis of nearly 100,000 undermethylated (or methylation filtration) maize sequences. Comparison with the rice genome reveals that methylation filtration results in a more comprehensive representation of maize genes than those that result from expressed sequence tags or transposon insertion sites sequences. About 7% of the repetitive DNA is unmethylated and thus selected in our libraries, but potentially active transposons and unmethylated organelle genomes can be identified. Reverse transcription polymerase chain reaction can be used to finish the maize transcriptome.

  17. Comparative Analysis of Genome and Epigenome in Closely Related Medaka Species Identifies Conserved Sequence Preferences for DNA Hypomethylated Domains.

    Science.gov (United States)

    Uno, Ayako; Nakamura, Ryohei; Tsukahara, Tatsuya; Qu, Wei; Sugano, Sumio; Suzuki, Yutaka; Morishita, Shinichi; Takeda, Hiroyuki

    2016-08-01

    The genomes of vertebrates are globally methylated, but a small portion of genomic regions are known to be hypomethylated. Although hypomethylated domains (HMDs) have been implicated in transcriptional regulation in various ways, how a HMD is determined in a particular genomic region remains elusive. To search for DNA motifs essential for the formation of HMDs, we performed the genome-wide comparative analysis of genome and DNA methylation patterns of the two medaka inbred lines, Hd-rRII1 and HNI-II, which are derived from northern and southern subpopulations of Japan and exhibit high levels of genetic variations (SNP, ∼ 3%). We successfully mapped > 70% of HMDs in both genomes and found that the majority of those mapped HMDs are conserved between the two lines (common HMDs). Unexpectedly, the average genetic variations are similar in the common HMD and other genome regions. However, we identified short well-conserved motifs that are specifically enriched in HMDs, suggesting that they may play roles in the establishment of HMDs in the medaka genome.

  18. Foundations for a syntatic pattern recognition system for genomic DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Searles, D.B.

    1993-03-01

    The goal of the proposed work is the creation of a software system that will perform sophisticated pattern recognition and related functions at a level of abstraction and with expressive power beyond current general-purpose pattern-matching systems for biological sequences; and with a more uniform language, environment, and graphical user interface, and with greater flexibility, extensibility, embeddability, and ability to incorporate other algorithms, than current special-purpose analytic software.

  19. Local repeat sequence organization of an intergenic spacer in the chloroplast genome of Chlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: a complex mode of “copy-choice replication”?

    Indian Academy of Sciences (India)

    Mahendra D Wagle; Subhojit Sen; Basuthkar J Rao

    2001-12-01

    Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA of Chlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt+) in a cross between a pair of polymorphic interfertile strains of Chlamydomonas (C. reinhardtii and C. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, ``P2” (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3′ end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, ``P2” seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely ``sequence-scrambled” product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a ``unique” new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a ``complex path” of copy-choice replication.

  20. Extracting biological knowledge from DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    De La Vega, F.M. [CINVESTAV-IPN (Mexico); Thieffry, D. [Universite Libre de Bruxelles, Rhode-Saint-Genese (Belgium)]|[Universidad Nacional Autonoma de Mexico, Morelos (Mexico); Collado-Vides, J. [Universidad Nacional Autonoma de Mexico, Morelos (Mexico)

    1996-12-31

    This session describes the elucidation of information from dna sequences and what challenges computational biologists face in their task of summarizing and deciphering the human genome. Techniques discussed include methods from statistics, information theory, artificial intelligence and linguistics. 1 ref.

  1. On the sequence-directed nature of human gene mutation: the role of genomic architecture and the local DNA sequence environment in mediating gene mutations underlying human inherited disease.

    Science.gov (United States)

    Cooper, David N; Bacolla, Albino; Férec, Claude; Vasquez, Karen M; Kehrer-Sawatzki, Hildegard; Chen, Jian-Min

    2011-10-01

    Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher order features of the genomic architecture. The human genome is now recognized to contain "pervasive architectural flaws" in that certain DNA sequences are inherently mutation prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here, we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of noncanonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair and may serve to increase mutation frequencies in generalized fashion (i.e., both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease. © 2011 Wiley-Liss, Inc.

  2. Mitochondrial DNA sequence evolution in shorebird populations.

    NARCIS (Netherlands)

    Wenink, P.W.

    1994-01-01

    This thesis describes the global molecular population structure of two shorebird species, in particular of the dunlin, Calidris alpina, by means of comparative sequence analysis of the most variable part of the mitochondrial DNA (mtDNA) genome. There are several reasons why mtDNA is the molecule of

  3. Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.

    Science.gov (United States)

    Fu, Chong-Yun; Liu, Wu-Ge; Liu, Di-Lin; Li, Ji-Hua; Zhu, Man-Shan; Liao, Yi-Long; Liu, Zhen-Rong; Zeng, Xue-Qin; Wang, Feng

    2016-03-01

    Next-generation sequencing technologies provide opportunities to further understand genetic variation, even within closely related cultivars. We performed whole genome resequencing of two elite indica rice varieties, RGD-7S and Taifeng B, whose F1 progeny showed hybrid weakness and hybrid vigor when grown in the early- and late-cropping seasons, respectively. Approximately 150 million 100-bp pair-end reads were generated, which covered ∼86% of the rice (Oryza sativa L. japonica 'Nipponbare') reference genome. A total of 2,758,740 polymorphic sites including 2,408,845 SNPs and 349,895 InDels were detected in RGD-7S and Taifeng B, respectively. Applying stringent parameters, we identified 961,791 SNPs and 46,640 InDels between RGD-7S and Taifeng B (RGD-7S/Taifeng B). The density of DNA polymorphisms was 256.8 SNPs and 12.5 InDels per 100 kb for RGD-7S/Taifeng B. Copy number variations (CNVs) were also investigated. In RGD-7S, 1989 of 2727 CNVs were overlapped in 218 genes, and 1231 of 2010 CNVs were annotated in 175 genes in Taifeng B. In addition, we verified a subset of InDels in the interval of hybrid weakness genes, Hw3 and Hw4, and obtained some polymorphic InDel markers, which will provide a sound foundation for cloning hybrid weakness genes. Analysis of genomic variations will also contribute to understanding the genetic basis of hybrid weakness and heterosis.

  4. Rhipicephalus (Boophilus) microplus strain Deutsch, whole genome shotgun sequencing project first submission of genome sequence

    Science.gov (United States)

    The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence difficult. Cot filtration/selection techniques were used to reduce the repetitive fraction of the tick genome and enrich for the fraction of DNA with gene-containing regions. The Cot-selected ...

  5. 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.

  6. DNA Sequencing Using capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Barry Karger

    2011-05-09

    The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linked polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other

  7. Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

    Science.gov (United States)

    Gouveia, Juceli Gonzalez; Wolf, Ivan Rodrigo; de Moraes-Manécolo, Vivian Patrícia Oliveira; Bardella, Vanessa Belline; Ferracin, Lara Munique; Giuliano-Caetano, Lucia; da Rosa, Renata; Dias, Ana Lúcia

    2016-12-01

    Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C0t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C0t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

  8. Pash 3.0: A versatile software package for read mapping and integrative analysis of genomic and epigenomic variation using massively parallel DNA sequencing

    Directory of Open Access Journals (Sweden)

    Chen Zuozhou

    2010-11-01

    Full Text Available Abstract Background Massively parallel sequencing readouts of epigenomic assays are enabling integrative genome-wide analyses of genomic and epigenomic variation. Pash 3.0 performs sequence comparison and read mapping and can be employed as a module within diverse configurable analysis pipelines, including ChIP-Seq and methylome mapping by whole-genome bisulfite sequencing. Results Pash 3.0 generally matches the accuracy and speed of niche programs for fast mapping of short reads, and exceeds their performance on longer reads generated by a new generation of massively parallel sequencing technologies. By exploiting longer read lengths, Pash 3.0 maps reads onto the large fraction of genomic DNA that contains repetitive elements and polymorphic sites, including indel polymorphisms. Conclusions We demonstrate the versatility of Pash 3.0 by analyzing the interaction between CpG methylation, CpG SNPs, and imprinting based on publicly available whole-genome shotgun bisulfite sequencing data. Pash 3.0 makes use of gapped k-mer alignment, a non-seed based comparison method, which is implemented using multi-positional hash tables. This allows Pash 3.0 to run on diverse hardware platforms, including individual computers with standard RAM capacity, multi-core hardware architectures and large clusters.

  9. The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences

    Directory of Open Access Journals (Sweden)

    Kalinowski Jörn

    2005-06-01

    Full Text Available Abstract Background The genus Corynebacterium includes Gram-positive microorganisms of great biotechnologically importance, such as Corynebacterium glutamicum and Corynebacterium efficiens, as well as serious human pathogens, such as Corynebacterium diphtheriae and Corynebacterium jeikeium. Although genome sequences of the respective species have been determined recently, the knowledge about the repertoire of transcriptional regulators and the architecture of global regulatory networks is scarce. Here, we apply a combination of bioinformatic tools and a comparative genomic approach to identify and characterize a set of conserved DNA-binding transcriptional regulators in the four corynebacterial genomes. Results A collection of 127 DNA-binding transcriptional regulators was identified in the C. glutamicum ATCC 13032 genome, whereas 103 regulators were detected in C. efficiens YS-314, 63 in C. diphtheriae NCTC 13129 and 55 in C. jeikeium K411. According to amino acid sequence similarities and protein structure predictions, the DNA-binding transcriptional regulators were grouped into 25 regulatory protein families. The common set of DNA-binding transcriptional regulators present in the four corynebacterial genomes consists of 28 proteins that are apparently involved in the regulation of cell division and septation, SOS and stress response, carbohydrate metabolism and macroelement and metal homeostasis. Conclusion This work describes characteristic features of a set of conserved DNA-binding transcriptional regulators present within the corynebacterial core genome. The knowledge on the physiological function of these proteins should not only contribute to our understanding of the regulation of gene expression but will also provide the basis for comprehensive modeling of transcriptional regulatory networks of these species.

  10. Analysis and validation of genome-specific DNA variations in 5' flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

    Institute of Scientific and Technical Information of China (English)

    LONG; Hai; WEI; Yuming

    2006-01-01

    The thirty-three 5' flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety 'Chinese Spring' was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5' flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5' flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.

  11. Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA

    DEFF Research Database (Denmark)

    Liu, Hongtai; Gao, Ya; Hu, Zhiyang

    2016-01-01

    through Maternal Plasma Sequencing (FCAPS) to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated. Results 33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results......Objectives The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT) for fetal copy number variants (CNVs) in clinical samples, using a whole-genome sequencing method. Method A total of 919 archived maternal plasma samples with karyotyping/microarray results......, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads) whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis...

  12. Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae: Unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer

    Directory of Open Access Journals (Sweden)

    Lemieux Claude

    2008-06-01

    Full Text Available Abstract Background To gain insight into the branching order of the five main lineages currently recognized in the green algal class Chlorophyceae and to expand our understanding of chloroplast genome evolution, we have undertaken the sequencing of chloroplast DNA (cpDNA from representative taxa. The complete cpDNA sequences previously reported for Chlamydomonas (Chlamydomonadales, Scenedesmus (Sphaeropleales, and Stigeoclonium (Chaetophorales revealed tremendous variability in their architecture, the retention of only few ancestral gene clusters, and derived clusters shared by Chlamydomonas and Scenedesmus. Unexpectedly, our recent phylogenies inferred from these cpDNAs and the partial sequences of three other chlorophycean cpDNAs disclosed two major clades, one uniting the Chlamydomonadales and Sphaeropleales (CS clade and the other uniting the Oedogoniales, Chaetophorales and Chaetopeltidales (OCC clade. Although molecular signatures provided strong support for this dichotomy and for the branching of the Oedogoniales as the earliest-diverging lineage of the OCC clade, more data are required to validate these phylogenies. We describe here the complete cpDNA sequence of Oedogonium cardiacum (Oedogoniales. Results Like its three chlorophycean homologues, the 196,547-bp Oedogonium chloroplast genome displays a distinctive architecture. This genome is one of the most compact among photosynthetic chlorophytes. It has an atypical quadripartite structure, is intron-rich (17 group I and 4 group II introns, and displays 99 different conserved genes and four long open reading frames (ORFs, three of which are clustered in the spacious inverted repeat of 35,493 bp. Intriguingly, two of these ORFs (int and dpoB revealed high similarities to genes not usually found in cpDNA. At the gene content and gene order levels, the Oedogonium genome most closely resembles its Stigeoclonium counterpart. Characters shared by these chlorophyceans but missing in members

  13. Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution

    Directory of Open Access Journals (Sweden)

    Purves Joanne

    2012-09-01

    Full Text Available Abstract Background Staphylococcus aureus Repeat (STAR elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Results Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. Conclusions The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis.

  14. Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution.

    Science.gov (United States)

    Purves, Joanne; Blades, Matthew; Arafat, Yasrab; Malik, Salman A; Bayliss, Christopher D; Morrissey, Julie A

    2012-09-28

    Staphylococcus aureus Repeat (STAR) elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis.

  15. Sequencing and comparing whole mitochondrial genomes ofanimals

    Energy Technology Data Exchange (ETDEWEB)

    Boore, Jeffrey L.; Macey, J. Robert; Medina, Monica

    2005-04-22

    Comparing complete animal mitochondrial genome sequences is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. Not only are they much more informative than shorter sequences of individual genes for inferring evolutionary relatedness, but these data also provide sets of genome-level characters, such as the relative arrangements of genes, that can be especially powerful. We describe here the protocols commonly used for physically isolating mtDNA, for amplifying these by PCR or RCA, for cloning,sequencing, assembly, validation, and gene annotation, and for comparing both sequences and gene arrangements. On several topics, we offer general observations based on our experiences to date with determining and comparing complete mtDNA sequences.

  16. Large scale full-length cDNA sequencing reveals a unique genomic landscape in a lepidopteran model insect, Bombyx mori.

    Science.gov (United States)

    Suetsugu, Yoshitaka; Futahashi, Ryo; Kanamori, Hiroyuki; Kadono-Okuda, Keiko; Sasanuma, Shun-ichi; Narukawa, Junko; Ajimura, Masahiro; Jouraku, Akiya; Namiki, Nobukazu; Shimomura, Michihiko; Sezutsu, Hideki; Osanai-Futahashi, Mizuko; Suzuki, Masataka G; Daimon, Takaaki; Shinoda, Tetsuro; Taniai, Kiyoko; Asaoka, Kiyoshi; Niwa, Ryusuke; Kawaoka, Shinpei; Katsuma, Susumu; Tamura, Toshiki; Noda, Hiroaki; Kasahara, Masahiro; Sugano, Sumio; Suzuki, Yutaka; Fujiwara, Haruhiko; Kataoka, Hiroshi; Arunkumar, Kallare P; Tomar, Archana; Nagaraju, Javaregowda; Goldsmith, Marian R; Feng, Qili; Xia, Qingyou; Yamamoto, Kimiko; Shimada, Toru; Mita, Kazuei

    2013-09-01

    The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

  17. Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray generated from the shotgun libraries previously used in the sequencing of this bacterial genome

    Directory of Open Access Journals (Sweden)

    Zaini Paulo A

    2010-05-01

    Full Text Available Abstract Background From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC, clones that were representative of the largest possible number of coding sequences (CDSs were selected to create a DNA microarray platform on glass slides (XACarray. The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.

  18. The complete chloroplast genome sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene organization and a biased distribution of genes on the two DNA strands

    Directory of Open Access Journals (Sweden)

    Lemieux Claude

    2006-04-01

    Full Text Available Abstract Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. While the basal position of the Prasinophyceae is well established, the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae (UTC remains uncertain. The five complete chloroplast DNA (cpDNA sequences currently available for representatives of these classes display considerable variability in overall structure, gene content, gene density, intron content and gene order. Among these genomes, that of the chlorophycean green alga Chlamydomonas reinhardtii has retained the least ancestral features. The two single-copy regions, which are separated from one another by the large inverted repeat (IR, have similar sizes, rather than unequal sizes, and differ radically in both gene contents and gene organizations relative to the single-copy regions of prasinophyte and ulvophyte cpDNAs. To gain insights into the various changes that underwent the chloroplast genome during the evolution of chlorophycean green algae, we have sequenced the cpDNA of Scenedesmus obliquus, a member of a distinct chlorophycean lineage. Results The 161,452 bp IR-containing genome of Scenedesmus features single-copy regions of similar sizes, encodes 96 genes, i.e. only two additional genes (infA and rpl12 relative to its Chlamydomonas homologue and contains seven group I and two group II introns. It is clearly more compact than the four UTC algal cpDNAs that have been examined so far, displays the lowest proportion of short repeats among these algae and shows a stronger bias in clustering of genes on the same DNA strand compared to Chlamydomonas cpDNA. Like the latter genome, Scenedesmus cpDNA displays only a few ancestral gene clusters. The two chlorophycean genomes share 11 gene clusters that are not found in previously sequenced trebouxiophyte and ulvophyte cpDNAs as well as a few genes that have an unusual structure; however, their single

  19. Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA.

    Directory of Open Access Journals (Sweden)

    Hongtai Liu

    Full Text Available The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT for fetal copy number variants (CNVs in clinical samples, using a whole-genome sequencing method.A total of 919 archived maternal plasma samples with karyotyping/microarray results, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated.33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results in the study. Ten false positive results and two false negative results were obtained. The sensitivity and specificity of detection deletions/duplications were 84.21% and 98.42%, respectively.Whole-genome sequencing-based NIPT has high performance in detecting genome-wide CNVs, in particular >10Mb CNVs using the current FCAPS algorithm. It is possible to implement the current method in NIPT to prenatally screening for fetal CNVs.

  20. DNA sequence recognition by hybridization to short oligomers : experimental verification of the method on the E-coli genome.

    Energy Technology Data Exchange (ETDEWEB)

    Milosavljevic, A.; Savkovic, S.; Crkvenjakov, R.; Salbego, D.; Serrato, H.; Kreuzer, H.; Gemmell, A.; Batus, S.; Grujic, D.; Carnahan, S.; Tepavcevic, J.; Center for Mechanistic Biology and Biotechnology

    1996-10-01

    A newly developed method for sequence recognition by hybridization to short oligomers is verified for the first time in genome-scale experiments. The experiments involved hybridization of 15,328 randomly selected 2-kb genomic clones of Escherichia coli with 997 short oligomer probes to detect complementary oligomers within the clones. Lists of oligomers detected within individual clones were compiled into a database. The database was then searched using known E. coli sequences as queries. The goal was to recognize the clones that are identical or similar to the query sequences. A total of 76 putative recognitions were tested in two separate but complementary recognition experiments. The results indicate high specificity of recognition. Current and prospective applications of this novel method are discussed.

  1. DNA sequence recognition by hybridization to short oligomers: experimental verification of the method on the E. coli genome.

    Science.gov (United States)

    Milosavljević, A; Savković, S; Crkvenjakov, R; Salbego, D; Serrato, H; Kreuzer, H; Gemmell, A; Batus, S; Grujić, D; Carnahan, S; Paunesku, T; Tepavcević, J

    1996-10-01

    A newly developed method for sequence recognition by hybridization to short oligomers is verified for the first time in genome-scale experiments. The experiments involved hybridization of 15,328 randomly selected 2-kb genomic clones of Escherichia coli with 997 short oligomer probes to detect complementary oligomers within the clones. Lists of oligomers detected within individual clones were compiled into a database. The database was then searched using known E. coli sequences as queries. The goal was to recognize the clones that are identical or similar to the query sequences. A total of 76 putative recognitions were tested in two separate but complementary recognition experiments. The results indicate high specificity of recognition. Current and prospective applications of this novel method are discussed.

  2. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    Directory of Open Access Journals (Sweden)

    Farkhondeh Saba

    2017-01-01

    Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

  3. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    Directory of Open Access Journals (Sweden)

    Farkhondeh Saba

    2016-09-01

    Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

  4. Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1.

    Science.gov (United States)

    Isegawa, Y; Sheng, J; Sokawa, Y; Yamanishi, K; Nakagomi, O; Ueda, S

    1992-12-01

    A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Construction of genome-wide physical BAC contigs using mapped cDNA as probes: Toward an integrated BAC library resource for genome sequencing and analysis. Annual report, July 1995--January 1997

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, S.C.; Bocskai, D.; Cao, Y. [and others

    1997-12-31

    The goal of human genome project is to characterize and sequence entire genomes of human and several model organisms, thus providing complete sets of information on the entire structure of transcribed, regulatory and other functional regions for these organisms. In the past years, a number of useful genetic and physical markers on human and mouse genomes have been made available along with the advent of BAC library resources for these organisms. The advances in technology and resource development made it feasible to efficiently construct genome-wide physical BAC contigs for human and other genomes. Currently, over 30,000 mapped STSs and 27,000 mapped Unigenes are available for human genome mapping. ESTs and cDNAs are excellent resources for building contig maps for two reasons. Firstly, they exist in two alternative forms--as both sequence information for PCR primer pairs, and cDoreen genomic libraries efficiently for large number of DNA probes by combining over 100 cDNA probes in each hybridization. Second, the linkage and order of genes are rather conserved among human, mouse and other model organisms. Therefore, gene markers have advantages over random anonymous STSs in building maps for comparative genomic studies.

  6. Improved rapid amplification of cDNA ends (RACE) for mapping both the 5' and 3' terminal sequences of paramyxovirus genomes.

    Science.gov (United States)

    Li, Zhuo; Yu, Meng; Zhang, Hong; Wang, Hai-Yan; Wang, Lin-Fa

    2005-12-01

    Rapid amplification of cDNA ends (RACE) is a powerful PCR-based technique for determination of RNA terminal sequences. However, most of the RACE methods reported in the literature are developed specifically for the mapping of eukaryotic transcripts with 3' poly-A tail and 5' cap structure. In this study, an improved RACE strategy was developed which allows both 5' and 3' RACE of paramyxovirus genomic RNA using the same set of common molecular biology reagents without having to rely on expensive RACE kits. Mapping of RNA genome terminal sequences is an essential part of characterizing novel paramyxoviruses since these sequences contain important signals for genome replication and transcription, and are important molecular markers for studying virus evolution. The usefulness of this strategy was demonstrated by rapid characterization of both genome ends for a novel paramyxovirus recently isolated from human kidney primary cells. The RACE strategy described in this paper is simple, cost-effective and can be used to map genome ends of any RNA viruses.

  7. Lineage-specific variations of congruent evolution among DNA sequences from three genomes, and relaxed selective constraints on rbcL in Cryptomonas (Cryptophyceae

    Directory of Open Access Journals (Sweden)

    Tran Hoang-Dung

    2005-10-01

    Full Text Available Abstract Background Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbcL genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbcL genes, and to compare the rbcL gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA, and nucleomorph SSU rDNA. Results Largely congruent branching patterns and accelerated evolutionary rates were found in nucleomorph SSU rDNA and rbcL genes in a clade that consisted of photosynthetic and colorless species suggesting a coevolution of the two genomes. The extremely accelerated rates in the rbcL phylogeny correlated with a shift from selection to mutation drift in codon usage of two-fold degenerate NNY codons comprising the amino acids asparagine, aspartate, histidine, phenylalanine, and tyrosine. Cysteine was the sole exception. The shift in codon usage seemed to follow a gradient from early diverging photosynthetic to late diverging photosynthetic or heterotrophic taxa along the branches. In the early branching taxa, codon preferences were changed in one to two amino acids, whereas in the late diverging taxa, including the colorless strains, between four and five amino acids showed changes in codon usage. Conclusion Nucleomorph and plastid gene phylogenies indicate that loss of photosynthesis in the colorless Cryptomonas strains examined in this study possibly was the result of accelerated evolutionary rates that started already in photosynthetic ancestors. Shifts in codon usage are usually considered to be caused by changes in functional

  8. Karyotype divergence and spreading of 5S rDNA sequences between genomes of two species: darter and emerald gobies ( Ctenogobius , Gobiidae).

    Science.gov (United States)

    Lima-Filho, P A; Bertollo, L A C; Cioffi, M B; Costa, G W W F; Molina, W F

    2014-01-01

    Karyotype analyses of the cryptobenthic marine species Ctenogobius boleosoma and C. smaragdus were performed by means of classical and molecular cytogenetics, including physical mapping of the multigene 18S and 5S rDNA families. C. boleosoma has 2n = 44 chromosomes (2 submetacentrics + 42 acrocentrics; FN = 46) with a single chromosome pair each carrying 18S and 5S ribosomal sites; whereas C. smaragdus has 2n = 48 chromosomes (2 submetacentrics + 46 acrocentrics; FN = 50), also with a single pair bearing 18S rDNA, but an extensive increase in the number of GC-rich 5S rDNA sites in 21 chromosome pairs. The highly divergent karyotypes among Ctenogobius species contrast with observations in several other marine fish groups, demonstrating an accelerated rate of chromosomal evolution mediated by both chromosomal rearrangements and the extensive dispersion of 5S rDNA sequences in the genome. © 2014 S. Karger AG, Basel.

  9. A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

    Science.gov (United States)

    Pu, Szu-Yuan; Wu, Ren-Huang; Tsai, Ming-Han; Yang, Chi-Chen; Chang, Chung-Ming; Yueh, Andrew

    2014-07-01

    Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses. © 2014 The Authors.

  10. 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...

  11. Mitochondrial DNA sequence evolution in shorebird populations

    NARCIS (Netherlands)

    Wenink, P.W.

    1994-01-01

    This thesis describes the global molecular population structure of two shorebird species, in particular of the dunlin, Calidris alpina, by means of comparative sequence analysis of the most variable part of the mitochondrial DNA (mtDNA) genome. There are several reasons

  12. The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

    Directory of Open Access Journals (Sweden)

    Lemieux Claude

    2006-02-01

    Full Text Available Abstract Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae, in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR featuring an inverted rRNA operon and a small single-copy (SSC region containing 14 genes normally found in the large single-copy (LSC region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage. Results The 151,933 bp IR-containing genome of Oltmannsiellopsis differs considerably from Pseudendoclonium and other chlorophyte cpDNAs in intron content and gene order, but shares close similarities with its ulvophyte homologue at the levels of quadripartite architecture, gene content and gene density. Oltmannsiellopsis cpDNA encodes 105 genes, contains five group I introns, and features many short dispersed repeats. As in Pseudendoclonium cpDNA, the rRNA genes in the IR are transcribed toward the single copy region featuring the genes typically found in the ancestral LSC region, and the opposite single copy region harbours genes characteristic of both the ancestral SSC and LSC regions. The 52 genes that were transferred from the ancestral LSC to SSC region include 12 of those observed in Pseudendoclonium cpDNA. Surprisingly, the overall gene organization of

  13. Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: Lactobacillus casei.

    Science.gov (United States)

    Scornec, Hélène; Tichit, Magali; Bouchier, Christiane; Pédron, Thierry; Cavin, Jean-François; Sansonetti, Philippe J; Licandro-Seraut, Hélène

    2014-11-01

    Random transposon mutagenesis followed by adequate screening methods is an unavoidable procedure to characterize genetics of bacterial adaptation to environmental changes. We have recently constructed a mutant library of Lactobacillus casei and we aimed to fully annotate it. However, we have observed that, for L. casei which is a difficult to lyse bacterium, methods used to identify the transposon insertion site in a few mutants (transposon rescue by restriction and recircularization or PCR-based methods) were not transposable for a larger number because they are too time-consuming and sometimes not reliable. Here, we describe a method for large-scale and reliable identification of transposon insertion sites in a L. casei mutant library of 9250 mutants. DNA extraction procedure based on silica membranes in 96-column format was optimized to obtain genomic DNA from a large number of mutants. Then reliable direct genomic sequencing was improved to fit the obtained genomic DNA extracts. Using this procedure, readable and identifiable sequences were obtained for 87% of the L. casei mutants. This method extends the applications of a library of this type, reduces the number of insertions needed to be screened, and allows selection of specific mutants from an arrayed and stored mutant library. This method is applicable to any already existing mutant library (obtained by transposon or insertional mutagenesis) and could be useful for other bacterial species, especially for highly lysis-resistant bacteria species such as lactic acid bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Extensive mitochondrial genome rearrangements between Cerithioidea and Hypsogastropoda (Mollusca; Caenogastropoda) as determined from the partial nucleotide sequences of the mitochondrial DNA of Cerithidea djadjariensis and Batillaria cumingi.

    Science.gov (United States)

    Kojima, Shigeaki

    2010-06-01

    Partial nucleotide sequences ( approximately 8000 bp) of the mitochondrial DNA of two cerithioidean gastropod species-Cerithidea djadjariensis and Batillaria cumingi-were determined. The order of mitochondrial genes (eight protein genes, two ribosomal RNA genes, and nine transfer RNA genes) was identical between these two species. and remarkably different from the previously reported order in other gastropods. The results indicate that the genome structure of the common ancestor of Cerithioidea and its sister group, Hypsogastropoda, is almost identical to that of the common ancestor of Gastropoda; moreover, independent mitochondrial genome rearrangements were identified between the lineages of Cerithioidea and Hypsogastropoda. The rearrangements within Cerithioidea can be explained by the inversion of a single tRNA gene, two translocations of a single tRNA gene, and three translocations of a genome fragment containing a tRNA gene and protein-coding gene(s).

  15. DNA Sequencing Using capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Barry Karger

    2011-05-09

    The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linked polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other

  16. [DNA sequencing technology and automatization of it].

    Science.gov (United States)

    Kraev, A S

    1991-01-01

    Precise manipulations with genetic material, typical for modern experiments in molecular biology and in new biotechnology, require a capability to determine DNA base sequence. This capability enables today to exploit specific genetic knowledge for the dissection of complex cell processes and for modulation of cell metabolism in transgenic organisms. The review focuses on such DNA sequencing technologies that are widespread in general laboratory practice. They can safely be called, with the availability of commercial reagents, industrial techniques. Modern DNA sequencing requires recurrent breakdown of large genomic DNA into smaller pieces, that are then amplified, sequenced and the initial long stretch reconstructed via overlap of small pieces. The DNA sequencing process has several steps: a DNA fragment is obtained in sufficient quantity and purity, it is converted to a form suitable for a particular sequencing method, a sequencing reaction is performed and its products fractionated; and finally the resultant data are interpreted (i.e. an autoradiograph is read into a computer memory) and a long sequence in reconstructed via overlap of short stretches. These steps are considered in separate parts; an accent is made on sequencing strategies with respect to their biological task. In the last part, possibilities for automation of sequencing experiment are considered, followed by a discussion of domestic problems in DNA sequencing.

  17. Identification of ancient remains through genomic sequencing

    Science.gov (United States)

    Blow, Matthew J.; Zhang, Tao; Woyke, Tanja; Speller, Camilla F.; Krivoshapkin, Andrei; Yang, Dongya Y.; Derevianko, Anatoly; Rubin, Edward M.

    2008-01-01

    Studies of ancient DNA have been hindered by the preciousness of remains, the small quantities of undamaged DNA accessible, and the limitations associated with conventional PCR amplification. In these studies, we developed and applied a genomewide adapter-mediated emulsion PCR amplification protocol for ancient mammalian samples estimated to be between 45,000 and 69,000 yr old. Using 454 Life Sciences (Roche) and Illumina sequencing (formerly Solexa sequencing) technologies, we examined over 100 megabases of DNA from amplified extracts, revealing unbiased sequence coverage with substantial amounts of nonredundant nuclear sequences from the sample sources and negligible levels of human contamination. We consistently recorded over 500-fold increases, such that nanogram quantities of starting material could be amplified to microgram quantities. Application of our protocol to a 50,000-yr-old uncharacterized bone sample that was unsuccessful in mitochondrial PCR provided sufficient nuclear sequences for comparison with extant mammals and subsequent phylogenetic classification of the remains. The combined use of emulsion PCR amplification and high-throughput sequencing allows for the generation of large quantities of DNA sequence data from ancient remains. Using such techniques, even small amounts of ancient remains with low levels of endogenous DNA preservation may yield substantial quantities of nuclear DNA, enabling novel applications of ancient DNA genomics to the investigation of extinct phyla. PMID:18426903

  18. Sequenciamento de DNA de nova geração e suas aplicações na genômica de plantas Next generation DNA sequencing and its applications in plant genomics

    Directory of Open Access Journals (Sweden)

    Mayra Costa da Cruz Gallo de Carvalho

    2010-03-01

    Full Text Available As plataformas de sequenciamento de nova geração são uma alternativa poderosa para estudos de genômica estrutural e funcional. Na genômica de plantas, os trabalhos com as novas plataformas têm sido destinados ao sequenciamento de transcritos, ressequenciamento ou sequenciamento de novo de genomas plastidiais. Neste trabalho, são detalhadas as tecnologias das plataformas mais utilizadas atualmente, bem como é revisada a aplicação dessas tecnologias na genômica estrutural e funcional de plantas.The next-generation DNA sequencing technologies are a powerful alternative to studies in structural and functional genomics. In plant genomics studies, the work with these new platforms has been used for the sequencing of transcripts, re-sequencing, and the de novo sequencing of plastid genomes. This research details the technological principles of the next-generation DNA sequencing platforms most used and reviews its application in structural and functional plant genomics.

  19. Simple sequence repeats in mycobacterial genomes

    Indian Academy of Sciences (India)

    Vattipally B Sreenu; Pankaj Kumar; Javaregowda Nagaraju; Hampapathalu A Nagarajaram

    2007-01-01

    Simple sequence repeats (SSRs) or microsatellites are the repetitive nucleotide sequences of motifs of length 1–6 bp. They are scattered throughout the genomes of all the known organisms ranging from viruses to eukaryotes. Microsatellites undergo mutations in the form of insertions and deletions (INDELS) of their repeat units with some bias towards insertions that lead to microsatellite tract expansion. Although prokaryotic genomes derive some plasticity due to microsatellite mutations they have in-built mechanisms to arrest undue expansions of microsatellites and one such mechanism is constituted by post-replicative DNA repair enzymes MutL, MutH and MutS. The mycobacterial genomes lack these enzymes and as a null hypothesis one could expect these genomes to harbour many long tracts. It is therefore interesting to analyse the mycobacterial genomes for distribution and abundance of microsatellites tracts and to look for potentially polymorphic microsatellites. Available mycobacterial genomes, Mycobacterium avium, M. leprae, M. bovis and the two strains of M. tuberculosis (CDC1551 and H37Rv) were analysed for frequencies and abundance of SSRs. Our analysis revealed that the SSRs are distributed throughout the mycobacterial genomes at an average of 220–230 SSR tracts per kb. All the mycobacterial genomes contain few regions that are conspicuously denser or poorer in microsatellites compared to their expected genome averages. The genomes distinctly show scarcity of long microsatellites despite the absence of a post-replicative DNA repair system. Such severe scarcity of long microsatellites could arise as a result of strong selection pressures operating against long and unstable sequences although influence of GC-content and role of point mutations in arresting microsatellite expansions can not be ruled out. Nonetheless, the long tracts occasionally found in coding as well as non-coding regions may account for limited genome plasticity in these genomes.

  20. Second generation DNA sequencing of the mitogenome of the Chinstrap penguin and comparative genomics of Antarctic penguins.

    Science.gov (United States)

    Subramanian, Sankar; Lingala, Syamala Gowri; Swaminathan, Siva; Huynen, Leon; Lambert, David

    2014-08-01

    The complete mitochondrial genome of the Chinstrap penguin (Pygoscelis antarcticus) was sequenced and compared with other penguin mitogenomes. The genome is 15,972 bp in length with the number and order of protein coding genes and RNAs being very similar to that of other known penguin mitogenomes. Comparative nucleotide analysis showed the Chinstrap mitogenome shares 94% homology with the mitogenome of its sister species, Pygoscelis adelie (Adélie penguin). Divergence at nonsynonymous nucleotide positions was found to be up to 23 times less than that observed in synonymous positions of protein coding genes, suggesting high selection constraints. The complete mitogenome data will be useful for genetic and evolutionary studies of penguins.

  1. The first determination of DNA sequence of a specific gene.

    Science.gov (United States)

    Inouye, Masayori

    2016-05-10

    How and when the first DNA sequence of a gene was determined? In 1977, F. Sanger came up with an innovative technology to sequence DNA by using chain terminators, and determined the entire DNA sequence of the 5375-base genome of bacteriophage φX 174 (Sanger et al., 1977). While this Sanger's achievement has been recognized as the first DNA sequencing of genes, we had determined DNA sequence of a gene, albeit a partial sequence, 11 years before the Sanger's DNA sequence (Okada et al., 1966).

  2. No genome barriers to promiscuous DNA

    Science.gov (United States)

    Lewin, R.

    1984-06-01

    Farrelly and Butow (1983) used the term 'promiscuous DNA' in their report of the apparent natural transfer of yeast mitochondrial DNA sequences into the nuclear genome. Ellis (1982) applied the same term in an editorial comment. It is pointed out since that time the subject of DNA's promiscuity has exploded with a series of reports. According to a report by Stern (1984), movement of DNA sequences between chloroplasts and mitochondria is not just a rare event but is a rampant process. It was recently concluded that 'the widespread presence of ctDNA sequences in plant mtDNA is best regarded as a dramatic demonstration of the dynamo nature of interactions between the chloroplast and the mitochondrion, similar to the ongoing process of interorganellar DNA transfer already documented between mitochondrion and nucleus and between chloroplast and nucleus'.

  3. Towards clinical molecular diagnosis of inherited cardiac conditions: a comparison of bench-top genome DNA sequencers.

    Directory of Open Access Journals (Sweden)

    Xinzhong Li

    Full Text Available Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations.We evaluated two next-generation sequencing (NGS platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm, and sequenced on the MiSeq (Illumina and Ion Torrent PGM (Life Technologies. Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%. At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs, with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS.MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias

  4. Sequence determination of cDNA clones of transcripts from the tumor-associated region of the Marek's disease virus genome.

    Science.gov (United States)

    Iwata, A; Ueda, S; Ishihama, A; Hirai, K

    1992-04-01

    The number of 132-bp tandem direct repeats within the long inverted repeat region of the Marek's disease virus type 1 (MDV1) genome increases concomitantly with the loss of oncogenicity during serial passages in cultured cells. Twelve clones carrying the 132-bp sequence were isolated from a cDNA library constructed from chicken embryo fibroblasts infected with the MDV1 Md5 strain. Through sequence analysis of a cDNA clone and primer extension analysis, the corresponding mRNA was found to be a linear transcript which included the two 132-bp tandem direct repeats. Two open reading frames were found in this transcript. One had a week homology with v-fms. The other should increase its size concomitantly with expansion of the 132-bp tandem direct repeat. PCR analysis of both cDNA clones and RNA gave amplified products which were as large as that produced from the genomic clone, indicating that a majority of mRNA from this region is composed of unspliced transcripts.

  5. Cyanophage Pf-WMP4, a T7-like phage infecting the freshwater cyanobacterium Phormidium foveolarum: complete genome sequence and DNA translocation.

    Science.gov (United States)

    Liu, Xinyao; Shi, Miao; Kong, Shuanglei; Gao, Yin; An, Chengcai

    2007-09-15

    We report the complete 40,938-bp genome sequence of a cyanophage, Pf-WMP4, which infects the freshwater cyanobacterium Phormidium foveolarum Gom. Nine of the forty-five potential open reading frames in the Pf-WMP4 genome share similarities with the genes found in T7-like phages. Using in vitro transcription, we found that seven promoters at the leftmost end of the genome can be recognized by the host RNA polymerase. By blocking transcriptional and translational inhibitors, we found that Pf-WMP4 DNA translocation, with an average translocation rate of 19.8+/-2.7 bp s(-1) at 28 degrees C, requires both host transcription and protein synthesis of an unknown factor. Therefore the mechanism of cyanophage Pf-WMP4 DNA injection may be driven both by a T7-like internalization mechanism as well as an additional unknown mechanism requiring de novo protein synthesis. Our analysis of the Pf-WMP4 genome sheds new light on the translocation strategies and evolutionary traces of phages belonging to the T7 supergroup.

  6. The complete DNA sequence of vaccinia virus.

    Science.gov (United States)

    Goebel, S J; Johnson, G P; Perkus, M E; Davis, S W; Winslow, J P; Paoletti, E

    1990-11-01

    The complete DNA sequence of the genome of vaccinia virus has been determined. The genome consisted of 191,636 bp with a base composition of 66.6% A + T. We have identified 198 "major" protein-coding regions and 65 overlapping "minor" regions, for a total of 263 potential genes. Genes encoded by the virus were located by examination of DNA sequence characteristics and compared with existing vaccinia virus mapping analyses, sequence data, and transcription data. These genes were found to be compactly organized along the genome with relatively few regions of noncoding sequences. Whereas several similarities to proteins of known function were discerned, the function of the majority of proteins encoded by these open reading frames is as yet undetermined.

  7. gargammel: a sequence simulator for ancient DNA.

    Science.gov (United States)

    Renaud, Gabriel; Hanghøj, Kristian; Willerslev, Eske; Orlando, Ludovic

    2016-10-29

    Ancient DNA has emerged as a remarkable tool to infer the history of extinct species and past populations. However, many of its characteristics, such as extensive fragmentation, damage and contamination, can influence downstream analyses. To help investigators measure how these could impact their analyses in silico, we have developed gargammel, a package that simulates ancient DNA fragments given a set of known reference genomes. Our package simulates the entire molecular process from post-mortem DNA fragmentation and DNA damage to experimental sequencing errors, and reproduces most common bias observed in ancient DNA datasets.

  8. Information Theory of DNA Sequencing

    CERN Document Server

    Motahari, Abolfazl; Tse, David

    2012-01-01

    DNA sequencing is the basic workhorse of modern day biology and medicine. Shotgun sequencing is the dominant technique used: many randomly located short fragments called reads are extracted from the DNA sequence, and these reads are assembled to reconstruct the original sequence. By drawing an analogy between the DNA sequencing problem and the classic communication problem, we define an information theoretic notion of sequencing capacity. This is the maximum number of DNA base pairs that can be resolved reliably per read, and provides a fundamental limit to the performance that can be achieved by any assembly algorithm. We compute the sequencing capacity explicitly for a simple statistical model of the DNA sequence and the read process. Using this framework, we also study the impact of noise in the read process on the sequencing capacity.

  9. Improved genome sequencing using an engineered transposase.

    Science.gov (United States)

    Kia, Amirali; Gloeckner, Christian; Osothprarop, Trina; Gormley, Niall; Bomati, Erin; Stephenson, Michelle; Goryshin, Igor; He, Molly Min

    2017-01-17

    Next-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours. When compared to the traditional ligation-based methods, transposed-based Nextera has a slight insertion bias. Here we present the discovery of a mutant transposase (Tn5-059) with a lowered GC insertion bias through protein engineering. We demonstrate Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. We also observe higher library diversity generated by Tn5-059 when compared to Nextera v2 for human exomes, which leads to less sequencing and lower cost per genome. In addition, when used for human exomes, Tn5-059 delivers consistent library insert size over a range of input DNA, allowing up to a tenfold variance from the 50 ng input recommendation. Enhanced DNA input tolerance of Tn5-059 can translate to flexibility and robustness of workflow. DNA input tolerance together with superior uniformity of coverage and lower AT dropouts extend the applications of transposase based library preps. We discuss possible mechanisms of improvements in Tn5-059, and potential advantages of using the new mutant in varieties of applications including microbiome sequencing and chromatin profiling.

  10. Levenshtein error-correcting barcodes for multiplexed DNA sequencing

    NARCIS (Netherlands)

    Buschmann, Tilo; Bystrykh, Leonid V.

    2013-01-01

    Background: High-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multi

  11. An Efficient Strategy Developed for Next-Generation Sequencing of Endosymbiont Genomes Performed Using Crude DNA Isolated from Host Tissues: A Case Study of Blattabacterium cuenoti Inhabiting the Fat Bodies of Cockroaches.

    Science.gov (United States)

    Kinjo, Yukihiro; Saitoh, Seikoh; Tokuda, Gaku

    2015-01-01

    Whole-genome sequencing has emerged as one of the most effective means to elucidate the biological roles and molecular features of obligate intracellular symbionts (endosymbionts). However, the de novo assembly of an endosymbiont genome remains a challenge when host and/or mitochondrial DNA sequences are present in a dataset and hinder the assembly of the genome. By focusing on the traits of genome evolution in endosymbionts, we herein developed and investigated a genome-assembly strategy that consisted of two consecutive procedures: the selection of endosymbiont contigs from an output obtained from a de novo assembly performed using a TBLASTX search against a reference genome, named TBLASTX Contig Selection and Filtering (TCSF), and the iterative reassembling of the genome from reads mapped on the selected contigs, named Iterative Mapping and ReAssembling (IMRA), to merge the contigs. In order to validate this approach, we sequenced two strains of the cockroach endosymbiont Blattabacterium cuenoti and applied this strategy to the datasets. TCSF was determined to be highly accurate and sensitive in contig selection even when the genome of a distantly related free-living bacterium was used as a reference genome. Furthermore, the use of IMRA markedly improved sequence assemblies: the genomic sequence of an endosymbiont was almost completed from a dataset containing only 3% of the sequences of the endosymbiont's genome. The efficiency of our strategy may facilitate further studies on endosymbionts.

  12. cDNA cloning and sequence analysis of genome segments S8 from rice black-streaked dwarf virus

    Institute of Scientific and Technical Information of China (English)

    张恒木; 陈剑平; 薛庆中; 雷娟利

    2002-01-01

    Genome segments S8 of two Chinese isolates of rice black-streaked dwarf virus (RBSDV), one from Zhejiang Province and another from Hebei Province, were amplified by RT-PCR and sequenced. Both segments consisted of 1936 nts in full length (EMBL accession numbers were AJ297431 and AJ297432, respectively) and contained only one big open reading frame which encoded a polypeptide with molecular weight of 68kD. The two Chinese isolates shared 94.0% and 96.5% identity at nucleotide and amino acid level, respectively. They shared 94.5-94.9% and 92.5-92.9% homology with S8 of RBSDV Japanese isolate at nucleotide and amino acid level, respectively; shared 85.1-87.6% and 91.7-91.9% homology with S7 of Italian MRDV (maize rough dwarf virus).

  13. Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Anita Kloss-Brandstätter

    Full Text Available Oral squamous cell carcinoma (OSCC is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA mutations in primary oral tumors, recurrences and metastases.We performed an in-depth validation of mtDNA next-generation sequencing (NGS on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base.We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.

  14. Whitefly (Bemisia tabaci genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous cDNA libraries

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

    Full Text Available Abstract Background The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae. Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. Results To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV and Tomato mottle virus (ToMoV. In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production

  15. From DNA Sequences to Chemical Structures – Methods for Mining Microbial Genomic and Metagenomic Data Sets for New Natural Products

    Directory of Open Access Journals (Sweden)

    Jurica Zucko

    2010-01-01

    Full Text Available Rapid mining of large genomic and metagenomic data sets for modular polyketide synthases, non-ribosomal peptide synthetases and hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene clusters has been achieved using the generic computer program packages ClustScan and CompGen. These program packages perform the annotation with the hierarchical structuring into polypeptides, modules and domains, as well as storage and graphical presentations of the data. This aims to achieve the most accurate predictions of the activities and specificities of catalytically active domains that can be made with present knowledge, leading to a prediction of the most likely chemical structures produced by these enzymes. The program packages also allow generation of novel clusters by homologous recombination of the annotated genes in silico. ClustScan and CompGen were used to construct a custom database of known compounds (CSDB and of predicted entirely novel recombinant products (r-CSDB that can be used for in silico screening with computer aided drug design technology. The use of these programs has been exemplified by analysing genomic sequences from terrestrial prokaryotes and eukaryotic microorganisms, a marine metagenomic data set and a newly discovered example of a 'shared metabolic pathway' in marine-microbial endosymbiosis.

  16. Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

    Science.gov (United States)

    Sun, Hengyi; Zang, Xiaonan; Liu, Yuantao; Cao, Xiaofei; Wu, Fei; Huang, Xiaoyun; Jiang, Minjie; Zhang, Xuecheng

    2015-12-01

    Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

  17. Biosensors for DNA sequence detection

    Science.gov (United States)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  18. Viral genome sequencing by random priming methods

    Directory of Open Access Journals (Sweden)

    Zhang Xinsheng

    2008-01-01

    Full Text Available Abstract Background Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing. Results We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000–15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter. Conclusion The method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.

  19. Genomic Sequence Comparisons, 1987-2003 Final Report

    Energy Technology Data Exchange (ETDEWEB)

    George M. Church

    2004-07-29

    This project was to develop new DNA sequencing and RNA and protein quantitation methods and related genome annotation tools. The project began in 1987 with the development of multiplex sequencing (published in Science in 1988), and one of the first automated sequencing methods. This lead to the first commercial genome sequence in 1994 and to the establishment of the main commercial participants (GTC then Agencourt) in the public DOE/NIH genome project. In collaboration with GTC we contributed to one of the first complete DOE genome sequences, in 1997, that of Methanobacterium thermoautotropicum, a species of great relevance to energy-rich gas production.

  20. Visible periodicity of strong nucleosome DNA sequences.

    Science.gov (United States)

    Salih, Bilal; Tripathi, Vijay; Trifonov, Edward N

    2015-01-01

    Fifteen years ago, Lowary and Widom assembled nucleosomes on synthetic random sequence DNA molecules, selected the strongest nucleosomes and discovered that the TA dinucleotides in these strong nucleosome sequences often appear at 10-11 bases from one another or at distances which are multiples of this period. We repeated this experiment computationally, on large ensembles of natural genomic sequences, by selecting the strongest nucleosomes--i.e. those with such distances between like-named dinucleotides, multiples of 10.4 bases, the structural and sequence period of nucleosome DNA. The analysis confirmed the periodicity of TA dinucleotides in the strong nucleosomes, and revealed as well other periodic sequence elements, notably classical AA and TT dinucleotides. The matrices of DNA bendability and their simple linear forms--nucleosome positioning motifs--are calculated from the strong nucleosome DNA sequences. The motifs are in full accord with nucleosome positioning sequences derived earlier, thus confirming that the new technique, indeed, detects strong nucleosomes. Species- and isochore-specific variations of the matrices and of the positioning motifs are demonstrated. The strong nucleosome DNA sequences manifest the highest hitherto nucleosome positioning sequence signals, showing the dinucleotide periodicities in directly observable rather than in hidden form.

  1. Nucleotide sequence and transcript organization of a region of the vaccinia virus genome which encodes a constitutively expressed gene required for DNA replication.

    Science.gov (United States)

    Roseman, N A; Hruby, D E

    1987-05-01

    A vaccinia virus (VV) gene required for DNA replication has been mapped to the left side of the 16-kilobase (kb) VV HindIII D DNA fragment by marker rescue of a DNA- temperature-sensitive mutant, ts17, using cloned fragments of the viral genome. The region of VV DNA containing the ts17 locus (3.6 kb) was sequenced. This nucleotide sequence contains one complete open reading frame (ORF) and two incomplete ORFs reading from left to right. Analysis of this region at early times revealed that transcription from the incomplete upstream ORF terminates coincidentally with the complete ORF encoding the ts17 gene product, which is directly downstream. The predicted proteins encoded by this region correlate well with polypeptides mapped by in vitro translation of hybrid-selected early mRNA. The nucleotide sequences of a 1.3-kb BglII fragment derived from ts17 and from two ts17 revertants were also determined, and the nature of the ts17 mutation was identified. S1 nuclease protection studies were carried out to determine the 5' and 3' ends of the transcripts and to examine the kinetics of expression of the ts17 gene during viral infection. The ts17 transcript is present at both early and late times postinfection, indicating that this gene is constitutively expressed. Surprisingly, the transcriptional start throughout infection occurs at the proposed late regulatory element TAA, which immediately precedes the putative initiation codon ATG. Although the biological activity of the ts17-encoded polypeptide was not identified, it was noted that in ts17-infected cells, expression of a nonlinked VV immediate-early gene (thymidine kinase) was deregulated at the nonpermissive temperature. This result may indicate that the ts17 gene product is functionally required at an early step of the VV replicative cycle.

  2. Graphene nanodevices for DNA sequencing

    Science.gov (United States)

    Heerema, Stephanie J.; Dekker, Cees

    2016-02-01

    Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.

  3. Bisprimer--a program for the design of primers for bisulfite-based genomic sequencing of both plant and Mammalian DNA samples.

    Science.gov (United States)

    Kovacova, Viera; Janousek, Bohuslav

    2012-01-01

    Plants and animals differ in the sequence context of the methylated sites in DNA. Plants exhibit cytosine methylation in CG, CHG, and CHH sites, whereas CG methylation is the only form present in mammals (with an exception of the early embryonic development). This fact must be taken into account in the design of primers for bisulfite-based genomic sequencing because CHG and CHH sites can remain unmodified. Surprisingly, no user-friendly primer design program is publicly available that could be used to design primers in plants and to simultaneously check the properties of primers such as the potential for primer-dimer formation. For studies concentrating on particular DNA loci, the correct design of primers is crucial. The program, called BisPrimer, includes 2 different subprograms for the primer design, the first one for mammals and the second one for angiosperm plants. Each subprogram is divided into 2 variants. The first variant serves to design primers that preferentially bind to the bisulfite-modified primer-binding sites (C to U conversion). This type of primer preferentially amplifies the bisulfite-converted DNA strands. This feature can help to avoid problems connected with an incomplete bisulfite modification that can sometimes occur for technical reasons. The second variant is intended for the analysis of samples that are supposed to consist of a mixture of DNA molecules that have different levels of cytosine methylation (e.g., pollen DNA). In this case, the aim is to minimize the selection in favor of either less methylated or more methylated molecules.

  4. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    National Research Council Canada - National Science Library

    Farkhondeh Saba; Moslem Papizadeh; Javad Khansha; Mahshid Sedghi; Mehrnoosh Rasooli; Mohammad Ali Amoozegar; Mohammad Reza Soudi; Seyed Abolhassan Shahzadeh Fazeli

    2017-01-01

    Background: Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction...

  5. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    National Research Council Canada - National Science Library

    Farkhondeh Saba; Moslem Papizadeh; Javad Khansha; Mahshid Sedghi; Mehrnoosh Rasooli; Mohammad Ali Amoozegar; Mohammad Reza Soudi; Seyed Abolhassan Shahzadeh Fazeli

    2016-01-01

    Background: Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction...

  6. Identifying driver mutations in sequenced cancer genomes

    DEFF Research Database (Denmark)

    Raphael, Benjamin J; Dobson, Jason R; Oesper, Layla

    2014-01-01

    High-throughput DNA sequencing is revolutionizing the study of cancer and enabling the measurement of the somatic mutations that drive cancer development. However, the resulting sequencing datasets are large and complex, obscuring the clinically important mutations in a background of errors, noise......, and random mutations. Here, we review computational approaches to identify somatic mutations in cancer genome sequences and to distinguish the driver mutations that are responsible for cancer from random, passenger mutations. First, we describe approaches to detect somatic mutations from high-throughput DNA...... sequencing data, particularly for tumor samples that comprise heterogeneous populations of cells. Next, we review computational approaches that aim to predict driver mutations according to their frequency of occurrence in a cohort of samples, or according to their predicted functional impact on protein...

  7. An approach to sequence DNA without tagging

    Science.gov (United States)

    Niu, Sanjun; Saraf, Ravi F.

    2002-10-01

    Microarray technology is playing an increasingly important role in biology and medicine and its application to genomics for gene expression analysis has already reached the market with a variety of commercially available instruments. In these combinatorial analysis methods, known probe single-strand DNA (ssDNA) 'primers' are attached in clusters of typically 100 µm × 100 µm pixels. Each pixel of the array has a slightly different sequence. On exposure to 'unknown' target ssDNA, the pixels with the right complementary probe ssDNA sequence convert to double-stranded DNA (dsDNA) by a hybridization reaction. To transduct the conversion of the pixel to dsDNA, the target ssDNA is labelled with a photoluminescent tag during the polymerase chain reaction (PCR) amplification process. Due to the statistical distribution of the tags in the target ssDNA, it becomes significantly difficult to implement these methods as a diagnostic tool in a pathology laboratory. A method to sequence DNA without tagging the molecule is developed. The fabrication process is compatible with current microelectronics and (emerging) soft-material fabrication technologies, allowing the method to be integrable with micro-electromechanical systems (MEMS) and lab-on-a-chip devices. An estimated sensitivity of 10-12 g on a 1 cm2 device area is obtained.

  8. The DNA sequence specificity of bleomycin cleavage in a systematically altered DNA sequence.

    Science.gov (United States)

    Gautam, Shweta D; Chen, Jon K; Murray, Vincent

    2017-08-01

    Bleomycin is an anti-tumour agent that is clinically used to treat several types of cancers. Bleomycin cleaves DNA at specific DNA sequences and recent genome-wide DNA sequencing specificity data indicated that the sequence 5'-RTGT*AY (where T* is the site of bleomycin cleavage, R is G/A and Y is T/C) is preferentially cleaved by bleomycin in human cells. Based on this DNA sequence, we constructed a plasmid clone to explore this bleomycin cleavage preference. By systematic variation of single nucleotides in the 5'-RTGT*AY sequence, we were able to investigate the effect of nucleotide changes on bleomycin cleavage efficiency. We observed that the preferred consensus DNA sequence for bleomycin cleavage in the plasmid clone was 5'-YYGT*AW (where W is A/T). The most highly cleaved sequence was 5'-TCGT*AT and, in fact, the seven most highly cleaved sequences conformed to the consensus sequence 5'-YYGT*AW. A comparison with genome-wide results was also performed and while the core sequence was similar in both environments, the surrounding nucleotides were different.

  9. Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

    Energy Technology Data Exchange (ETDEWEB)

    McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L.; dePamphilis, Claude W.

    2005-08-26

    Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.

  10. Genome-scale sequence data processing and epigenetic analysis of DNA methylation%基因组规模DNA 甲基化测序数据处理及其表观遗传分析

    Institute of Scientific and Technical Information of China (English)

    王庭璋; 单杲; 徐建红; 薛庆中

    2013-01-01

    鉴定DNA 甲基化胞嘧啶(mC)并能制作基因组规模甲基化图谱的新方法--BS-Seq,最近已被开发,它是基于新一代高通量测序结合DNA 亚硫酸氢盐转换技术,不仅可以从基因组规模洞察不同生物之间在DNA 甲基化水平和模式上的差异,也能从不同基因组区域,包括基因、外显子、重复序列等方面,阐明DNA 甲基化环境和核苷酸偏好上的保守性,加深理解DNA 胞嘧啶(C)甲基化在调控基因表达和沉默转座子等重复序列中所起的表观遗传学影响.文章举例介绍了DNA 甲基化位点数据预处理的具体步骤,通过处理分别将参考序列中的胞嘧啶(C)替换成胸腺嘧啶(T),鸟嘌呤(G)替换成腺嘌呤(A),而将读序列中的胞嘧啶(C)替换为胸腺嘧啶(T).文章综述了全基因组DNA 甲基化分析的主要内容,包括:(1)不同序列环境下的胞嘧啶甲基化; (2)全基因组上的甲基化的分布情况; (3)DNA 甲基化环境和核苷酸的偏好; (4)DNA-蛋白质互作位点上的DNA 甲基化; (5)不同基因结构元件的胞嘧啶甲基化程度.DNA 甲基化分析技术为研究不同物种的表观基因组,环境和表观互作提供了强大的工具,并为进一步发展人体疾病诊断和治疗方法提供理论基础.%A new approach recently developed for detecting cytosine DNA methylation (mC) and analyzing the genome-scale DNA methylation profiling, is called BS-Seq which is based on bisulfite conversion of genomic DNA combined with next-generation sequencing. The method can not only provide an insight into the difference of genome-scale DNA methylation among different organisms, but also reveal the conservation of DNA methylation in all contexts and nucleotide preference for different genomic regions, including genes, exons, and repetitive DNA sequences. It will be helpful to under- stand the epigenetic impacts of cytosine DNA methylation on the regulation of gene expression and maintaining silence of repetitive

  11. 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

  12. Female-specific DNA sequences in geese.

    Science.gov (United States)

    Huang, M C; Lin, W C; Horng, Y M; Rouvier, R; Huang, C W

    2003-07-01

    1. The OPAE random primers (Operon Technologies, Inc., CA) were used for random amplified polymorphic DNA (RAPD) fingerprinting in Chinese, White Roman and Landaise geese. One of these primers, OPAE-06, produced a 938-bp sex-specific fragment in all females and in no males of Chinese geese only. 2. A novel female-specific DNA sequence in Chinese goose was cloned and sequenced. Two primers, CGSex-F and CGSex-R, were designed in order to amplify a 912-bp sex-specific polymerase chain reaction (PCR) fragment on genomic DNA from female geese. 3. It was shown that a simple and effective PCR-based sexing technique could be used in the three goose breeds studied. 4. Nucleotide sequencing of the sex-specific fragments in White Roman and Landaise geese was performed and sequence differences were observed among these three breeds.

  13. Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

    Directory of Open Access Journals (Sweden)

    Holt Robert A

    2010-04-01

    Full Text Available Abstract Background Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar, but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. Results From existing expressed sequence tag (EST resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. Conclusions 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.

  14. Brief Guide to Genomics: DNA, Genes and Genomes

    Science.gov (United States)

    ... Breve guía de genómica A Brief Guide to Genomics DNA, Genes and Genomes Deoxyribonucleic acid (DNA) is ... genetic basis for health and disease. Implications of Genomics for Medical Science Virtually every human ailment has ...

  15. Detection of Non-Amplified Genomic DNA

    CERN Document Server

    Corradini, Roberto

    2012-01-01

    This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...

  16. Profiling genome-wide DNA methylation.

    Science.gov (United States)

    Yong, Wai-Shin; Hsu, Fei-Man; Chen, Pao-Yang

    2016-01-01

    DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

  17. Unexpected cross-species contamination in genome sequencing projects

    Directory of Open Access Journals (Sweden)

    Samier Merchant

    2014-11-01

    Full Text Available The raw data from a genome sequencing project sometimes contains DNA from contaminating organisms, which may be introduced during sample collection or sequence preparation. In some instances, these contaminants remain in the sequence even after assembly and deposition of the genome into public databases. As a result, searches of these databases may yield erroneous and confusing results. We used efficient microbiome analysis software to scan the draft assembly of domestic cow, Bos taurus, and identify 173 small contigs that appeared to derive from microbial contaminants. In the course of verifying these findings, we discovered that one genome, Neisseria gonorrhoeae TCDC-NG08107, although putatively a complete genome, contained multiple sequences that actually derived from the cow and sheep genomes. Our findings illustrate the need to carefully validate findings of anomalous DNA that rely on comparisons to either draft or finished genomes.

  18. Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

    Science.gov (United States)

    Nakano, Kazuma; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Minami, Maiko; Nakanishi, Tetsuhiro; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

    2017-07-01

    PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II's sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

  19. Isolation of Plasmodium falciparum by flow-cytometry: implications for single-trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival samples

    Directory of Open Access Journals (Sweden)

    Boissière Anne

    2012-05-01

    Full Text Available Abstract Background Flow cytometry and cell sorting are powerful tools enabling the selection of particular cell types within heterogeneous cell mixtures. These techniques, combined with whole genome amplification that non-specifically amplify small amounts of starting DNA, offer exciting new opportunities for the study of malaria genetics. Among them, two are tested in this paper: (1 single cell genotyping and (2 parasite DNA purification for subsequent whole genome sequencing using shotgun technologies. Methods The method described allows isolation of Plasmodium falciparum trophozoites, genotyping and whole genome sequencing from the blood of infected patients. For trophozoite isolation, parasite and host nuclei are stained using propidium iodide (PI followed by flow cytometry and cell sorting to separate trophozoites from host cells. Before genotyping or sequencing, whole genome amplification is used to increase the amount of DNA within sorted samples. The method has been specifically designed to deal with frozen blood samples. Results and conclusion The results demonstrate that single trophozoite genotyping is possible and that cell sorting can be successfully applied to reduce the contaminating host DNA for subsequent whole genome sequencing of parasites extracted from infected blood samples.

  20. [Mapping and human genome sequence program].

    Science.gov (United States)

    Weissenbach, J

    1997-03-01

    Until recently, human genome programs focused primarily on establishing maps that would provide signposts to researchers seeking to identify genes responsible for inherited diseases, as well as a basis for genome sequencing studies. Preestablished gene mapping goals have been reached. The over 7,000 microsatellite markers identified to date provide a map of sufficient density to allow localization of the gene of a monogenic disease with a precision of 1 to 2 million base pairs. The physical map, based on systematically arranged overlapping sets of artificial yeast chromosomes (YACs), has also made considerable headway during the last few years. The most recently published map covers more than 90% of the genome. However, currently available physical maps cannot be used for sequencing studies because multiple rearrangements occur in YACs. The recently developed sets of radioinduced hybrids are extremely useful for incorporating genes into existing maps. A network of American and European laboratories has successfully used these radioinduced hybrids to map 15,000 gene tags from large-scale cDNA library sequencing programs. There are increasingly pressing reasons for initiating large scale human genome sequencing studies.

  1. Two genome sequences of the same bacterial strain, Gluconacetobacter diazotrophicus PAl 5, suggest a new standard in genome sequence submission.

    Science.gov (United States)

    Giongo, Adriana; Tyler, Heather L; Zipperer, Ursula N; Triplett, Eric W

    2010-06-15

    Gluconacetobacter diazotrophicus PAl 5 is of agricultural significance due to its ability to provide fixed nitrogen to plants. Consequently, its genome sequence has been eagerly anticipated to enhance understanding of endophytic nitrogen fixation. Two groups have sequenced the PAl 5 genome from the same source (ATCC 49037), though the resulting sequences contain a surprisingly high number of differences. Therefore, an optical map of PAl 5 was constructed in order to determine which genome assembly more closely resembles the chromosomal DNA by aligning each sequence against a physical map of the genome. While one sequence aligned very well, over 98% of the second sequence contained numerous rearrangements. The many differences observed between these two genome sequences could be owing to either assembly errors or rapid evolutionary divergence. The extent of the differences derived from sequence assembly errors could be assessed if the raw sequencing reads were provided by both genome centers at the time of genome sequence submission. Hence, a new genome sequence standard is proposed whereby the investigator supplies the raw reads along with the closed sequence so that the community can make more accurate judgments on whether differences observed in a single stain may be of biological origin or are simply caused by differences in genome assembly procedures.

  2. Preparation of genomic DNA from bacteria.

    Science.gov (United States)

    Andreou, Lefkothea-Vasiliki

    2013-01-01

    The purpose of this protocol is the isolation of bulk cellular DNA from bacteria (alternatively see Preparation of genomic DNA from Saccharomyces cerevisiae or Isolation of Genomic DNA from Mammalian Cells protocols). Copyright © 2013 Elsevier Inc. All rights reserved.

  3. New stopping criteria for segmenting DNA sequences

    CERN Document Server

    Li, W

    2001-01-01

    We propose a solution on the stopping criterion in segmenting inhomogeneous DNA sequences with complex statistical patterns. This new stopping criterion is based on Bayesian Information Criterion (BIC) in the model selection framework. When this stopping criterion is applied to a left telomere sequence of yeast Saccharomyces cerevisiae and the complete genome sequence of bacterium Escherichia coli, borders of biologically meaningful units were identified (e.g. subtelomeric units, replication origin, and replication terminus), and a more reasonable number of domains was obtained. We also introduce a measure called segmentation strength which can be used to control the delineation of large domains. The relationship between the average domain size and the threshold of segmentation strength is determined for several genome sequences.

  4. Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens.

    Directory of Open Access Journals (Sweden)

    Martijn Staats

    Full Text Available Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes, but at least generating vital comparative genomic data for testing (phylogenetic, demographic and genetic hypotheses, that become increasingly more

  5. Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens.

    Science.gov (United States)

    Staats, Martijn; Erkens, Roy H J; van de Vossenberg, Bart; Wieringa, Jan J; Kraaijeveld, Ken; Stielow, Benjamin; Geml, József; Richardson, James E; Bakker, Freek T

    2013-01-01

    Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal

  6. Bov-B long interspersed repeated DNA (LINE) sequences are present in Vipera ammodytes phospholipase A2 genes and in genomes of Viperidae snakes.

    Science.gov (United States)

    Kordis, D; Gubensek, F

    1997-06-15

    Ammodytin L is a myotoxic Ser49 phospholipase A2 (PLA2) homologue, which is tissue-specifically expressed in the venom glands of Vipera ammodytes. The complete DNA sequence of the gene and its 5' and 3' flanking regions has been determined. The gene consists of five exons separated by four introns. Comparative analysis of the ammodytin L and ammodytoxin C genes shows that all intron and flanking sequences are considerably more conserved (93-97%) than the mature protein-coding exons. The pattern of nucleotide substitutions in protein-coding exons is not random but occurs preferentially on the first and the second positions of codons, which suggests positive Darwinian evolution for a new function. An Ruminantia specific ART-2 retroposon, recently recognised as a 5'-truncated Bov-B long interspersed repeated DNA (LINE) sequence, was identified in the fourth intron of both genes. This result suggests that ammodytin L and ammodytoxin C genes are derived by duplication of a common ancestral gene. The phylogenetic distribution of Bov-B LINE among vertebrate classes shows that, besides the Ruminantia, it is limited to Viperidae snakes (Vipera ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3' end of Bov-B LINE in the Vipera ammodytes genome is between 62,000 and 75,000. The absence of Bov-B LINE at orthologous positions in other snake PLA2 genes indicates that its retrotransposition in the V. ammodytes PLA2 gene locus has occurred quite recently, about 5 My ago. The amplification of Bov-B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it may be a valuable phylogenetic marker. The neighbor-joining phylogenetic tree shows two clusters of truncated Bov-B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.

  7. DNA Sequence Determination by Hybridization: A Strategy for Efficient Large-Scale Sequencing

    Science.gov (United States)

    Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoddy, J.; Funkhouser, W. K.; Koop, B.; Hood, L.; Crkvenjakov, R.

    1993-06-01

    The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.

  8. BSMAP: whole genome bisulfite sequence MAPping program

    Directory of Open Access Journals (Sweden)

    Li Wei

    2009-07-01

    Full Text Available Abstract Background Bisulfite sequencing is a powerful technique to study DNA cytosine methylation. Bisulfite treatment followed by PCR amplification specifically converts unmethylated cytosines to thymine. Coupled with next generation sequencing technology, it is able to detect the methylation status of every cytosine in the genome. However, mapping high-throughput bisulfite reads to the reference genome remains a great challenge due to the increased searching space, reduced complexity of bisulfite sequence, asymmetric cytosine to thymine alignments, and multiple CpG heterogeneous methylation. Results We developed an efficient bisulfite reads mapping algorithm BSMAP to address the above issues. BSMAP combines genome hashing and bitwise masking to achieve fast and accurate bisulfite mapping. Compared with existing bisulfite mapping approaches, BSMAP is faster, more sensitive and more flexible. Conclusion BSMAP is the first general-purpose bisulfite mapping software. It is able to map high-throughput bisulfite reads at whole genome level with feasible memory and CPU usage. It is freely available under GPL v3 license at http://code.google.com/p/bsmap/.

  9. Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories.

    Science.gov (United States)

    Jäger, Anne C; Alvarez, Michelle L; Davis, Carey P; Guzmán, Ernesto; Han, Yonmee; Way, Lisa; Walichiewicz, Paulina; Silva, David; Pham, Nguyen; Caves, Glorianna; Bruand, Jocelyne; Schlesinger, Felix; Pond, Stephanie J K; Varlaro, Joe; Stephens, Kathryn M; Holt, Cydne L

    2017-05-01

    Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR

  10. Ribosomal DNA copy number loss and sequence variation in cancer.

    Science.gov (United States)

    Xu, Baoshan; Li, Hua; Perry, John M; Singh, Vijay Pratap; Unruh, Jay; Yu, Zulin; Zakari, Musinu; McDowell, William; Li, Linheng; Gerton, Jennifer L

    2017-06-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

  11. Duplication in DNA Sequences

    Science.gov (United States)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  12. Investigating the prehistory of Tungusic peoples of Siberia and the Amur-Ussuri region with complete mtDNA genome sequences and Y-chromosomal markers.

    Directory of Open Access Journals (Sweden)

    Ana T Duggan

    Full Text Available Evenks and Evens, Tungusic-speaking reindeer herders and hunter-gatherers, are spread over a wide area of northern Asia, whereas their linguistic relatives the Udegey, sedentary fishermen and hunter-gatherers, are settled to the south of the lower Amur River. The prehistory and relationships of these Tungusic peoples are as yet poorly investigated, especially with respect to their interactions with neighbouring populations. In this study, we analyse over 500 complete mtDNA genome sequences from nine different Evenk and even subgroups as well as their geographic neighbours from Siberia and their linguistic relatives the Udegey from the Amur-Ussuri region in order to investigate the prehistory of the Tungusic populations. These data are supplemented with analyses of Y-chromosomal haplogroups and STR haplotypes in the Evenks, Evens, and neighbouring Siberian populations. We demonstrate that whereas the North Tungusic Evenks and Evens show evidence of shared ancestry both in the maternal and in the paternal line, this signal has been attenuated by genetic drift and differential gene flow with neighbouring populations, with isolation by distance further shaping the maternal genepool of the Evens. The Udegey, in contrast, appear quite divergent from their linguistic relatives in the maternal line, with a mtDNA haplogroup composition characteristic of populations of the Amur-Ussuri region. Nevertheless, they show affinities with the Evenks, indicating that they might be the result of admixture between local Amur-Ussuri populations and Tungusic populations from the north.

  13. Genome Sequences of Eight Morphologically Diverse Alphaproteobacteria▿

    OpenAIRE

    Brown, Pamela J.B.; Kysela, David T.; Buechlein, Aaron; Hemmerich, Chris; Brun, Yves V

    2011-01-01

    The Alphaproteobacteriacomprise morphologically diverse bacteria, including many species of stalked bacteria. Here we announce the genome sequences of eight alphaproteobacteria, including the first genome sequences of species belonging to the genera Asticcacaulis, Hirschia, Hyphomicrobium, and Rhodomicrobium.

  14. Genome sequences of eight morphologically diverse Alphaproteobacteria.

    Science.gov (United States)

    Brown, Pamela J B; Kysela, David T; Buechlein, Aaron; Hemmerich, Chris; Brun, Yves V

    2011-09-01

    The Alphaproteobacteria comprise morphologically diverse bacteria, including many species of stalked bacteria. Here we announce the genome sequences of eight alphaproteobacteria, including the first genome sequences of species belonging to the genera Asticcacaulis, Hirschia, Hyphomicrobium, and Rhodomicrobium.

  15. Genome Sequences of Eight Morphologically Diverse Alphaproteobacteria▿

    Science.gov (United States)

    Brown, Pamela J. B.; Kysela, David T.; Buechlein, Aaron; Hemmerich, Chris; Brun, Yves V.

    2011-01-01

    The Alphaproteobacteriacomprise morphologically diverse bacteria, including many species of stalked bacteria. Here we announce the genome sequences of eight alphaproteobacteria, including the first genome sequences of species belonging to the genera Asticcacaulis, Hirschia, Hyphomicrobium, and Rhodomicrobium. PMID:21705585

  16. A DNA Structure-Based Bionic Wavelet Transform and Its Application to DNA Sequence Analysis

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2003-01-01

    Full Text Available DNA sequence analysis is of great significance for increasing our understanding of genomic functions. An important task facing us is the exploration of hidden structural information stored in the DNA sequence. This paper introduces a DNA structure-based adaptive wavelet transform (WT – the bionic wavelet transform (BWT – for DNA sequence analysis. The symbolic DNA sequence can be separated into four channels of indicator sequences. An adaptive symbol-to-number mapping, determined from the structural feature of the DNA sequence, was introduced into WT. It can adjust the weight value of each channel to maximise the useful energy distribution of the whole BWT output. The performance of the proposed BWT was examined by analysing synthetic and real DNA sequences. Results show that BWT performs better than traditional WT in presenting greater energy distribution. This new BWT method should be useful for the detection of the latent structural features in future DNA sequence analysis.

  17. Structural variation in the chicken genome identified by paired-end next-generation DNA sequencing of reduced representation libraries

    NARCIS (Netherlands)

    Kerstens, H.H.D.; Crooijmans, R.P.M.A.; Dibbits, B.W.; Vereijken, A.; Okimoto, R.; Groenen, M.A.M.

    2011-01-01

    Background Variation within individual genomes ranges from single nucleotide polymorphisms (SNPs) to kilobase, and even megabase, sized structural variants (SVs), such as deletions, insertions, inversions, and more complex rearrangements. Although much is known about the extent of SVs in humans and

  18. Genomic Sequencing of Single Microbial Cells from Environmental Samples

    Energy Technology Data Exchange (ETDEWEB)

    Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark; Lasken, Roger S.

    2008-02-01

    Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

  19. COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.

    Science.gov (United States)

    Castellanos-Rizaldos, Elena; Milbury, Coren A; Karatza, Elli; Chen, Clark C; Makrigiorgos, G Mike; Merewood, Anne

    2014-01-01

    Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.

  20. Reconstructing cancer genomes from paired-end sequencing data

    Directory of Open Access Journals (Sweden)

    Oesper Layla

    2012-04-01

    Full Text Available Abstract Background A cancer genome is derived from the germline genome through a series of somatic mutations. Somatic structural variants - including duplications, deletions, inversions, translocations, and other rearrangements - result in a cancer genome that is a scrambling of intervals, or "blocks" of the germline genome sequence. We present an efficient algorithm for reconstructing the block organization of a cancer genome from paired-end DNA sequencing data. Results By aligning paired reads from a cancer genome - and a matched germline genome, if available - to the human reference genome, we derive: (i a partition of the reference genome into intervals; (ii adjacencies between these intervals in the cancer genome; (iii an estimated copy number for each interval. We formulate the Copy Number and Adjacency Genome Reconstruction Problem of determining the cancer genome as a sequence of the derived intervals that is consistent with the measured adjacencies and copy numbers. We design an efficient algorithm, called Paired-end Reconstruction of Genome Organization (PREGO, to solve this problem by reducing it to an optimization problem on an interval-adjacency graph constructed from the data. The solution to the optimization problem results in an Eulerian graph, containing an alternating Eulerian tour that corresponds to a cancer genome that is consistent with the sequencing data. We apply our algorithm to five ovarian cancer genomes that were sequenced as part of The Cancer Genome Atlas. We identify numerous rearrangements, or structural variants, in these genomes, analyze reciprocal vs. non-reciprocal rearrangements, and identify rearrangements consistent with known mechanisms of duplication such as tandem duplications and breakage/fusion/bridge (B/F/B cycles. Conclusions We demonstrate that PREGO efficiently identifies complex and biologically relevant rearrangements in cancer genome sequencing data. An implementation of the PREGO algorithm is

  1. Whole-genome amplification of single-cell genomes for next-generation sequencing.

    Science.gov (United States)

    Korfhage, Christian; Fisch, Evelyn; Fricke, Evelyn; Baedker, Silke; Loeffert, Dirk

    2013-10-11

    DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS), microarrays, or real-time PCR is often limited by the small amount of sample available. A single cell contains only one to four copies of the genomic DNA, depending on the organism (haploid or diploid organism) and the cell-cycle phase. The DNA content of a single cell ranges from a few femtograms in bacteria to picograms in mammalia. In contrast, a deep analysis of the genome currently requires a few hundred nanograms up to micrograms of genomic DNA for library formation necessary for NGS sequencing or labeling protocols (e.g., microarrays). Consequently, accurate whole-genome amplification (WGA) of single-cell DNA is required for reliable genetic analysis (e.g., NGS) and is particularly important when genomic DNA is limited. The use of single-cell WGA has enabled the analysis of genomic heterogeneity of individual cells (e.g., somatic genomic variation in tumor cells). This unit describes how the genome of single cells can be used for WGA for further genomic studies, such as NGS. Recommendations for isolation of single cells are given and common sources of errors are discussed.

  2. Perspectives of DNA microarray and next-generation DNA sequencing technologies

    Institute of Scientific and Technical Information of China (English)

    TENG XiaoKun; XIAO HuaSheng

    2009-01-01

    DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequenc-ing method was first introduced by the 454 Company in 2003, immediately followed by the establish-ment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.

  3. Plant DNA barcoding: from gene to genome.

    Science.gov (United States)

    Li, Xiwen; Yang, Yang; Henry, Robert J; Rossetto, Maurizio; Wang, Yitao; Chen, Shilin

    2015-02-01

    DNA barcoding is currently a widely used and effective tool that enables rapid and accurate identification of plant species; however, none of the available loci work across all species. Because single-locus DNA barcodes lack adequate variations in closely related taxa, recent barcoding studies have placed high emphasis on the use of whole-chloroplast genome sequences which are now more readily available as a consequence of improving sequencing technologies. While chloroplast genome sequencing can already deliver a reliable barcode for accurate plant identification it is not yet resource-effective and does not yet offer the speed of analysis provided by single-locus barcodes to unspecialized laboratory facilities. Here, we review the development of candidate barcodes and discuss the feasibility of using the chloroplast genome as a super-barcode. We advocate a new approach for DNA barcoding that, for selected groups of taxa, combines the best use of single-locus barcodes and super-barcodes for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely related plants at the species and population levels.

  4. DNA sequencing by nanopores: advances and challenges

    Science.gov (United States)

    Agah, Shaghayegh; Zheng, Ming; Pasquali, Matteo; Kolomeisky, Anatoly B.

    2016-10-01

    Developing inexpensive and simple DNA sequencing methods capable of detecting entire genomes in short periods of time could revolutionize the world of medicine and technology. It will also lead to major advances in our understanding of fundamental biological processes. It has been shown that nanopores have the ability of single-molecule sensing of various biological molecules rapidly and at a low cost. This has stimulated significant experimental efforts in developing DNA sequencing techniques by utilizing biological and artificial nanopores. In this review, we discuss recent progress in the nanopore sequencing field with a focus on the nature of nanopores and on sensing mechanisms during the translocation. Current challenges and alternative methods are also discussed.

  5. Synaptotagmin gene content of the sequenced genomes

    Directory of Open Access Journals (Sweden)

    Craxton Molly

    2004-07-01

    Full Text Available Abstract Background Synaptotagmins exist as a large gene family in mammals. There is much interest in the function of certain family members which act crucially in the regulated synaptic vesicle exocytosis required for efficient neurotransmission. Knowledge of the functions of other family members is relatively poor and the presence of Synaptotagmin genes in plants indicates a role for the family as a whole which is wider than neurotransmission. Identification of the Synaptotagmin genes within completely sequenced genomes can provide the entire Synaptotagmin gene complement of each sequenced organism. Defining the detailed structures of all the Synaptotagmin genes and their encoded products can provide a useful resource for functional studies and a deeper understanding of the evolution of the gene family. The current rapid increase in the number of sequenced genomes from different branches of the tree of life, together with the public deposition of evolutionarily diverse transcript sequences make such studies worthwhile. Results I have compiled a detailed list of the Synaptotagmin genes of Caenorhabditis, Anopheles, Drosophila, Ciona, Danio, Fugu, Mus, Homo, Arabidopsis and Oryza by examining genomic and transcript sequences from public sequence databases together with some transcript sequences obtained by cDNA library screening and RT-PCR. I have compared all of the genes and investigated the relationship between plant Synaptotagmins and their non-Synaptotagmin counterparts. Conclusions I have identified and compared 98 Synaptotagmin genes from 10 sequenced genomes. Detailed comparison of transcript sequences reveals abundant and complex variation in Synaptotagmin gene expression and indicates the presence of Synaptotagmin genes in all animals and land plants. Amino acid sequence comparisons indicate patterns of conservation and diversity in function. Phylogenetic analysis shows the origin of Synaptotagmins in multicellular eukaryotes and their

  6. Foundations for a syntatic pattern recognition system for genomic DNA sequences. [Annual] report, 1 December 1991--31 March 1993

    Energy Technology Data Exchange (ETDEWEB)

    Searles, D.B.

    1993-03-01

    The goal of the proposed work is the creation of a software system that will perform sophisticated pattern recognition and related functions at a level of abstraction and with expressive power beyond current general-purpose pattern-matching systems for biological sequences; and with a more uniform language, environment, and graphical user interface, and with greater flexibility, extensibility, embeddability, and ability to incorporate other algorithms, than current special-purpose analytic software.

  7. Enhanced Dynamic Algorithm of Genome Sequence Alignments

    Directory of Open Access Journals (Sweden)

    Arabi E. keshk

    2014-05-01

    Full Text Available The merging of biology and computer science has created a new field called computational biology that explore the capacities of computers to gain knowledge from biological data, bioinformatics. Computational biology is rooted in life sciences as well as computers, information sciences, and technologies. The main problem in computational biology is sequence alignment that is a way of arranging the sequences of DNA, RNA or protein to identify the region of similarity and relationship between sequences. This paper introduces an enhancement of dynamic algorithm of genome sequence alignment, which called EDAGSA. It is filling the three main diagonals without filling the entire matrix by the unused data. It gets the optimal solution with decreasing the execution time and therefore the performance is increased. To illustrate the effectiveness of optimizing the performance of the proposed algorithm, it is compared with the traditional methods such as Needleman-Wunsch, Smith-Waterman and longest common subsequence algorithms. Also, database is implemented for using the algorithm in multi-sequence alignments for searching the optimal sequence that matches the given sequence.

  8. Translational genomics for plant breeding with the genome sequence explosion.

    Science.gov (United States)

    Kang, Yang Jae; Lee, Taeyoung; Lee, Jayern; Shim, Sangrea; Jeong, Haneul; Satyawan, Dani; Kim, Moon Young; Lee, Suk-Ha

    2016-04-01

    The use of next-generation sequencers and advanced genotyping technologies has propelled the field of plant genomics in model crops and plants and enhanced the discovery of hidden bridges between genotypes and phenotypes. The newly generated reference sequences of unstudied minor plants can be annotated by the knowledge of model plants via translational genomics approaches. Here, we reviewed the strategies of translational genomics and suggested perspectives on the current databases of genomic resources and the database structures of translated information on the new genome. As a draft picture of phenotypic annotation, translational genomics on newly sequenced plants will provide valuable assistance for breeders and researchers who are interested in genetic studies.

  9. Sequencing and analysis of an Irish human genome.

    LENUS (Irish Health Repository)

    Tong, Pin

    2010-01-01

    Recent studies generating complete human sequences from Asian, African and European subgroups have revealed population-specific variation and disease susceptibility loci. Here, choosing a DNA sample from a population of interest due to its relative geographical isolation and genetic impact on further populations, we extend the above studies through the generation of 11-fold coverage of the first Irish human genome sequence.

  10. Slow DNA loss in the gigantic genomes of salamanders.

    Science.gov (United States)

    Sun, Cheng; López Arriaza, José R; Mueller, Rachel Lockridge

    2012-01-01

    Evolutionary changes in genome size result from the combined effects of mutation, natural selection, and genetic drift. Insertion and deletion mutations (indels) directly impact genome size by adding or removing sequences. Most species lose more DNA through small indels (i.e., ~1-30 bp) than they gain, which can result in genome reduction over time. Because this rate of DNA loss varies across species, small indel dynamics have been suggested to contribute to genome size evolution. Species with extremely large genomes provide interesting test cases for exploring the link between small indels and genome size; however, most large genomes remain relatively unexplored. Here, we examine rates of DNA loss in the tetrapods with the largest genomes-the salamanders. We used low-coverage genomic shotgun sequence data from four salamander species to examine patterns of insertion, deletion, and substitution in neutrally evolving non-long terminal repeat (LTR) retrotransposon sequences. For comparison, we estimated genome-wide DNA loss rates in non-LTR retrotransposon sequences from five other vertebrate genomes: Anolis carolinensis, Danio rerio, Gallus gallus, Homo sapiens, and Xenopus tropicalis. Our results show that salamanders have significantly lower rates of DNA loss than do other vertebrates. More specifically, salamanders experience lower numbers of deletions relative to insertions, and both deletions and insertions are skewed toward smaller sizes. On the basis of these patterns, we conclude that slow DNA loss contributes to genomic gigantism in salamanders. We also identify candidate molecular mechanisms underlying these differences and suggest that natural variation in indel dynamics provides a unique opportunity to study the basis of genome stability.

  11. DNA Sequence Alignment during Homologous Recombination.

    Science.gov (United States)

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination.

  12. DNA Sequence Alignment during Homologous Recombination*

    Science.gov (United States)

    Greene, Eric C.

    2016-01-01

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. PMID:27129270

  13. Structural Complexity of DNA Sequence

    Directory of Open Access Journals (Sweden)

    Cheng-Yuan Liou

    2013-01-01

    Full Text Available In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results.

  14. Non-invasive detection of genomic imbalances in Hodgkin/Reed-Sternberg cells in early and advanced stage Hodgkin's lymphoma by sequencing of circulating cell-free DNA: a technical proof-of-principle study.

    Science.gov (United States)

    Vandenberghe, Peter; Wlodarska, Iwona; Tousseyn, Thomas; Dehaspe, Luc; Dierickx, Daan; Verheecke, Magali; Uyttebroeck, Anne; Bechter, Oliver; Delforge, Michel; Vandecaveye, Vincent; Brison, Nathalie; Verhoef, Gregor E G; Legius, Eric; Amant, Frederic; Vermeesch, Joris R

    2015-02-01

    Hodgkin's lymphoma is one of the most common lymphoid neoplasms in young adults, but the low abundance of neoplastic Hodgkin/Reed-Sternberg cells in the tumour hampers the elucidation of its pathogenesis, biology, and diversity. After an incidental observation that genomic aberrations known to occur in Hodgkin's lymphoma were detectable in circulating cell-free DNA, this study was undertaken to investigate whether circulating cell-free DNA can be informative about genomic imbalances in Hodgkin's lymphoma. We applied massive parallel sequencing to circulating cell-free DNA in a prospective study of patients with biopsy proven nodular sclerosis Hodgkin's lymphoma. Genomic imbalances in Hodgkin/Reed-Sternberg cells were investigated by fluorescence in-situ hybridisation (FISH) on tumour specimens. By non-invasive prenatal testing, we observed several genomic imbalances in circulating cell-free DNA of a pregnant woman, who was subsequently diagnosed with early-stage nodular sclerosis Hodgkin's lymphoma stage IIA during gestation. FISH on tumour tissue confirmed corresponding genomic imbalances in Hodgkin/Reed-Sternberg cells. We prospectively studied circulating cell-free DNA of nine nodular sclerosis Hodgkin's lymphoma cases: eight at first diagnosis and one at first relapse. Seven patients had stage IIA disease and two had stage IVB disease. In eight, genomic imbalances were detected, including, among others, gain of chromosomes 2p and 9p, known to occur in Hodgkin's lymphoma. These gains and losses in circulating cell-free DNA were extensively validated by FISH on Hodgkin/Reed-Sternberg cells in biopsy samples. Initiation of chemotherapy induced normalisation of circulating cell-free DNA profiles within 2-6 weeks. The cell cycle indicator Ki67 and cleaved caspase-3 were detected in Hodgkin/Reed-Sternberg cells by immunohistochemistry, suggesting high turnover of Hodgkin/Reed-Sternberg cells. In early and advanced stage nodular sclerosis Hodgkin's lymphoma, genomic

  15. Oxford Nanopore MinION Sequencing and Genome Assembly

    Institute of Scientific and Technical Information of China (English)

    Hengyun Lu; Francesca Giordano; Zemin Ning

    2016-01-01

    The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology, led by Pacific Biosciences (PacBio), is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that pro-mises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies (ONT). MinION identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MinION has thus generated much excitement and interest in the geno-mics community. While de novo genome assemblies can be cheaply produced from SGS data, assem-bly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in gen-ome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.

  16. Fungal genome sequencing: basic biology to biotechnology.

    Science.gov (United States)

    Sharma, Krishna Kant

    2016-08-01

    The genome sequences provide a first glimpse into the genomic basis of the biological diversity of filamentous fungi and yeast. The genome sequence of the budding yeast, Saccharomyces cerevisiae, with a small genome size, unicellular growth, and rich history of genetic and molecular analyses was a milestone of early genomics in the 1990s. The subsequent completion of fission yeast, Schizosaccharomyces pombe and genetic model, Neurospora crassa initiated a revolution in the genomics of the fungal kingdom. In due course of time, a substantial number of fungal genomes have been sequenced and publicly released, representing the widest sampling of genomes from any eukaryotic kingdom. An ambitious genome-sequencing program provides a wealth of data on metabolic diversity within the fungal kingdom, thereby enhancing research into medical science, agriculture science, ecology, bioremediation, bioenergy, and the biotechnology industry. Fungal genomics have higher potential to positively affect human health, environmental health, and the planet's stored energy. With a significant increase in sequenced fungal genomes, the known diversity of genes encoding organic acids, antibiotics, enzymes, and their pathways has increased exponentially. Currently, over a hundred fungal genome sequences are publicly available; however, no inclusive review has been published. This review is an initiative to address the significance of the fungal genome-sequencing program and provides the road map for basic and applied research.

  17. Value of a newly sequenced bacterial genome

    DEFF Research Database (Denmark)

    Barbosa, Eudes; Aburjaile, Flavia F; Ramos, Rommel Tj

    2014-01-01

    and annotation will not be undertaken. It is important to know what is lost when we settle for a draft genome and to determine the "scientific value" of a newly sequenced genome. This review addresses the expected impact of newly sequenced genomes on antibacterial discovery and vaccinology. Also, it discusses...

  18. Insights from twenty years of bacterial genome sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Jun, Se Ran [ORNL; Nookaew, Intawat [ORNL; Leuze, Michael Rex [ORNL; Ahn, Tae-Hyuk [ORNL; Karpinets, Tatiana V [ORNL; Lund, Ole [Technical University of Denmark; Kora, Guruprasad H [ORNL; Wassenaar, Trudy [Molecular Microbiology & Genomics Consultants, Zotzenheim, Germany; Poudel, Suresh [ORNL; Ussery, David W [ORNL

    2015-01-01

    Since the first two complete bacterial genome sequences were published in 1995, the science of bacteria has dramatically changed. Using third-generation DNA sequencing, it is possible to completely sequence a bacterial genome in a few hours and identify some types of methylation sites along the genome as well. Sequencing of bacterial genome sequences is now a standard procedure, and the information from tens of thousands of bacterial genomes has had a major impact on our views of the bacterial world. In this review, we explore a series of questions to highlight some insights that comparative genomics has produced. To date, there are genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. However, the distribution is quite skewed towards a few phyla that contain model organisms. But the breadth is continuing to improve, with projects dedicated to filling in less characterized taxonomic groups. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides bacteria with immunity against viruses, which outnumber bacteria by tenfold. How fast can we go? Second-generation sequencing has produced a large number of draft genomes (close to 90 % of bacterial genomes in GenBank are currently not complete); third-generation sequencing can potentially produce a finished genome in a few hours, and at the same time provide methlylation sites along the entire chromosome. The diversity of bacterial communities is extensive as is evident from the genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. Genome sequencing can help in classifying an organism, and in the case where multiple genomes of the same species are available, it is possible to calculate the pan- and core genomes; comparison of more than 2000 Escherichia coli genomes finds an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Why do we care about bacterial genome

  19. Differentiation of Indica-Japonica rice revealed by insertion/deletion (InDel) fragments obtained from the comparative genomic study of DNA sequences between 93-11 (Indica) and Nipponbare (Japonica)

    Institute of Scientific and Technical Information of China (English)

    CAI Xingxing; LIU Jing; QIU Yinqiu; ZHAO Wei; SONG Zhiping; LU Baorong

    2007-01-01

    DNA polymorphisms from nucleotide insertion/deletions (InDels) in genomic sequences are the basis for developing InDel molecular markers.To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species,we studied 49 Indica,43 Japonica,and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs.Results indicated that of the 45 InDel primer pairs,41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%.The scatter plotting data of the principal component analysis (PCA) indicated that:(i) the InDel primer pairs can easily distinguish Indica from Japonica rice varieties,in addition to revealing their genetic differentiation;(ii) the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties;and (iii)the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types.It is concluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying lndica and Japonica rice varieties,and for studying genetic relationships of wild rice species,particularly in terms of the Indica-Japonica differentiation.

  20. Draft sequences of the radish (Raphanus sativus L.) genome.

    Science.gov (United States)

    Kitashiba, Hiroyasu; Li, Feng; Hirakawa, Hideki; Kawanabe, Takahiro; Zou, Zhongwei; Hasegawa, Yoichi; Tonosaki, Kaoru; Shirasawa, Sachiko; Fukushima, Aki; Yokoi, Shuji; Takahata, Yoshihito; Kakizaki, Tomohiro; Ishida, Masahiko; Okamoto, Shunsuke; Sakamoto, Koji; Shirasawa, Kenta; Tabata, Satoshi; Nishio, Takeshi

    2014-10-01

    Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥ 300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.

  1. 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.

  2. Perspectives of DNA microarray and next-generation DNA sequencing technologies

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research,in revealing both the structural and functional characteristics of genomes.In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics,systems biology and pharmacogenomics.The next-generation DNA sequencing method was first introduced by the 454 Company in 2003,immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies.Though it has not been long since the first emergence of this technology,with the fast and impressive improvement,the application of this technology has extended to almost all fields of genomics research,as a rival challenging the existing DNA microarray technology.This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research.

  3. Draft Genome Sequence of Lactobacillus rhamnosus 2166.

    OpenAIRE

    Karlyshev, Andrey V.; Melnikov, Vyacheslav G.; Kosarev, Igor V.; Abramov, Vyacheslav M.

    2014-01-01

    In this report, we present a draft sequence of the genome of Lactobacillus rhamnosus strain 2166, a potential novel probiotic. Genome annotation and read mapping onto a reference genome of L. rhamnosus strain GG allowed for the identification of the differences and similarities in the genomic contents and gene arrangements of these strains.

  4. Draft Genome Sequence of Lactobacillus rhamnosus 2166.

    OpenAIRE

    Karlyshev, Andrey V.; Melnikov, Vyacheslav G.; Kosarev, Igor V.; Abramov, Vyacheslav M.

    2014-01-01

    In this report, we present a draft sequence of the genome of Lactobacillus rhamnosus strain 2166, a potential novel probiotic. Genome annotation and read mapping onto a reference genome of L. rhamnosus strain GG allowed for the identification of the differences and similarities in the genomic contents and gene arrangements of these strains.

  5. Complete Genome Sequence of Human Respiratory Syncytial Virus from Lanzhou, China

    OpenAIRE

    Zhu, Chuanfeng; Fu, Shengfang; Zhou, Xv; Yu, Li

    2017-01-01

    ABSTRACT A complete genome of human respiratory syncytial virus was sequenced and analyzed. Phylogenetic analysis showed that the full-length human respiratory syncytial virus (HRSV) genome sequence belongs to gene type NA1. We sequenced the genome in order to create the full-length cDNA infectious clone and develop vaccines against HRSV.

  6. Draft Genome Sequence of "Terrisporobacter othiniensis" Isolated from a Blood Culture from a Human Patient

    DEFF Research Database (Denmark)

    Lund, Lars Christian; Sydenham, Thomas Vognbjerg; Høgh, Silje Vermedal

    2015-01-01

    "Terrisporobacter othiniensis" (proposed species) was isolated from a blood culture. Genomic DNA was sequenced using a MiSeq benchtop sequencer (Illumina) and assembled using the SPAdes genome assembler. This resulted in a draft genome sequence comprising 3,980,019 bp in 167 contigs containing 3...

  7. Scrutinizing virus genome termini by high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Shasha Li

    Full Text Available Analysis of genomic terminal sequences has been a major step in studies on viral DNA replication and packaging mechanisms. However, traditional methods to study genome termini are challenging due to the time-consuming protocols and their inefficiency where critical details are lost easily. Recent advances in next generation sequencing (NGS have enabled it to be a powerful tool to study genome termini. In this study, using NGS we sequenced one iridovirus genome and twenty phage genomes and confirmed for the first time that the high frequency sequences (HFSs found in the NGS reads are indeed the terminal sequences of viral genomes. Further, we established a criterion to distinguish the type of termini and the viral packaging mode. We also obtained additional terminal details such as terminal repeats, multi-termini, asymmetric termini. With this approach, we were able to simultaneously detect details of the genome termini as well as obtain the complete sequence of bacteriophage genomes. Theoretically, this application can be further extended to analyze larger and more complicated genomes of plant and animal viruses. This study proposed a novel and efficient method for research on viral replication, packaging, terminase activity, transcription regulation, and metabolism of the host cell.

  8. Value of a newly sequenced bacterial genome

    Institute of Scientific and Technical Information of China (English)

    Eudes; GV; Barbosa; Flavia; F; Aburjaile; Rommel; TJ; Ramos; Adriana; R; Carneiro; Yves; Le; Loir; Jan; Baumbach; Anderson; Miyoshi; Artur; Silva; Vasco; Azevedo

    2014-01-01

    Next-generation sequencing(NGS) technologies have made high-throughput sequencing available to medium- and small-size laboratories, culminating in a tidal wave of genomic information. The quantity of sequenced bacterial genomes has not only brought excitement to the field of genomics but also heightened expectations that NGS would boost antibacterial discovery and vaccine development. Although many possible drug and vaccine targets have been discovered, the success rate of genome-based analysis has remained below expectations. Furthermore, NGS has had consequences for genome quality, resulting in an exponential increase in draft(partial data) genome deposits in public databases. If no further interests are expressed for a particular bacterial genome, it is more likely that the sequencing of its genome will be limited to a draft stage, and the painstaking tasks of completing the sequencing of its genome and annotation will not be undertaken. It is important to know what is lost when we settle for a draft genome and to determine the "scientific value" of a newly sequenced genome. This review addresses the expected impact of newly sequenced genomes on antibacterial discovery and vaccinology. Also, it discusses the factors that could be leading to the increase in the number of draft deposits and the consequent loss of relevant biological information.

  9. Plasmodium knowlesi genome sequences from clinical isolates reveal extensive genomic dimorphism.

    Directory of Open Access Journals (Sweden)

    Miguel M Pinheiro

    Full Text Available Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and

  10. 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.

  11. Yeast DNA sequences initiating gene expression in Escherichia coli.

    Science.gov (United States)

    Lewin, Astrid; Tran, Thi Tuyen; Jacob, Daniela; Mayer, Martin; Freytag, Barbara; Appel, Bernd

    2004-01-01

    DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

  12. Local Renyi entropic profiles of DNA sequences

    Directory of Open Access Journals (Sweden)

    Vinga Susana

    2007-10-01

    Full Text Available Abstract Background In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM. Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs. Results The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at http://kdbio.inesc-id.pt/~svinga/ep/. Conclusion The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures.

  13. Preparing DNA libraries for multiplexed paired-end deep sequencing for Illumina GA sequencers.

    Science.gov (United States)

    Son, Mike S; Taylor, Ronald K

    2011-02-01

    Whole-genome sequencing, also known as deep sequencing, is becoming a more affordable and efficient way to identify SNP mutations, deletions, and insertions in DNA sequences across several different strains. Two major obstacles preventing the widespread use of deep sequencers are the costs involved in services used to prepare DNA libraries for sequencing and the overall accuracy of the sequencing data. This unit describes the preparation of DNA libraries for multiplexed paired-end sequencing using the Illumina GA series sequencer. Self-preparation of DNA libraries can help reduce overall expenses, especially if optimization is required for the different samples, and use of the Illumina GA Sequencer can improve the quality of the data.

  14. PCR primers for metazoan mitochondrial 12S ribosomal DNA sequences.

    Directory of Open Access Journals (Sweden)

    Ryuji J Machida

    Full Text Available BACKGROUND: Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. METHODOLOGY/PRINCIPAL FINDINGS: A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. CONCLUSIONS/SIGNIFICANCE: Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans.

  15. Pilot Sequencing of Onion Genomic DNA Reveals Fragments of Transposable Elements, Low Gene Densities, and Significant Gene Enrichment After Methyl Filtration

    Science.gov (United States)

    Onion (Allium cepa) is a diploid (2n=2x=16) monocot with one of the largest nuclear genomes among cultivated plants, over 6 and 16 times that of maize and rice, respectively. In this study, we sequenced onion BACs to estimate gene densities and investigate the nature and distribution of repetitive ...

  16. Genome-scale validation of deep-sequencing libraries.

    Directory of Open Access Journals (Sweden)

    Dominic Schmidt

    Full Text Available Chromatin immunoprecipitation followed by high-throughput (HTP sequencing (ChIP-seq is a powerful tool to establish protein-DNA interactions genome-wide. The primary limitation of its broad application at present is the often-limited access to sequencers. Here we report a protocol, Mab-seq, that generates genome-scale quality evaluations for nucleic acid libraries intended for deep-sequencing. We show how commercially available genomic microarrays can be used to maximize the efficiency of library creation and quickly generate reliable preliminary data on a chromosomal scale in advance of deep sequencing. We also exploit this technique to compare enriched regions identified using microarrays with those identified by sequencing, demonstrating that they agree on a core set of clearly identified enriched regions, while characterizing the additional enriched regions identifiable using HTP sequencing.

  17. From Sequence to Morphology - Long-Range Correlations in Complete Sequenced Genomes

    NARCIS (Netherlands)

    T.A. Knoch (Tobias)

    2004-01-01

    textabstractThe largely unresolved sequential organization, i.e. the relations within DNA sequences, and its connection to the three-dimensional organization of genomes was investigated by correlation analyses of completely sequenced chromosomes from Viroids, Archaea, Bacteria, Arabidopsis thali

  18. Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms.

    Science.gov (United States)

    Garzelli, Carlo; Lari, Nicoletta; Rindi, Laura

    2016-03-01

    The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes. MIRU-VNTR analysis revealed 47 unique patterns and 9 clusters including a total of 33 isolates (41% of total isolates); the relatively high proportion of Italian-born Beijing TB patients, often occurring in mixed clusters, supports the possibility of an ongoing cross-transmission of the Beijing genotype to autochthonous population. High rates of extra-pulmonary localization and drug-resistance, particularly MDR, frequently reported for Beijing strains in other settings, were not observed in our survey.

  19. [An intriguing model for 5S rDNA sequences dispersion in the genome of freshwater stingray Potamotrygon motoro (Chondrichthyes: Potamotrygonidae)].

    Science.gov (United States)

    Cruz, V P; Oliveira, C; Foresti, F

    2015-01-01

    5S rDNA genes of the stingray Potamotrygon motoro were PCR replicated, purified, cloned and sequenced. Two distinct classes of segments of different sizes were obtained. The smallest, with 342 bp units, was classified as class I, and the largest, with 1900 bp units, was designated as class II. Alignment with the consensus sequences for both classes showed changes in a few bases in the 5S rDNA genes. TATA-like sequences were detected in the nontranscribed spacer (NTS) regions of class I and a microsatellite (GCT) 10 sequence was detected in the NTS region of class II. The results obtained can help to understand the molecular organization of ribosomal genes and the mechanism of gene dispersion.

  20. Complete genome sequence of a new tobamovirus naturally infecting tomatoes in Mexico

    Science.gov (United States)

    The complete genomic sequence of a new tobamovirus in tomato was determined through deep sequencing and assembly of small RNAs, thenvalidated through Sanger sequencing of the overlapping RT-PCR products and rapid amplification of cDNA ends (RACE). Based on the genomic sequence identity (85%) to kn...

  1. Two-dimensional DNA displays for comparisons of bacterial genomes

    Directory of Open Access Journals (Sweden)

    Malloff Chad

    2003-01-01

    Full Text Available We have developed two whole genome-scanning techniques to aid in the discovery of polymorphisms as well as horizontally acquired genes in prokaryotic organisms. First, two-dimensional bacterial genomic display (2DBGD was developed using restriction enzyme fragmentation to separate genomic DNA based on size, and then employing denaturing gradient gel electrophoresis (DGGE in the second dimension to exploit differences in sequence composition. This technique was used to generate high-resolution displays that enable the direct comparison of > 800 genomic fragments simultaneously and can be adapted for the high-throughput comparison of bacterial genomes. 2DBGDs are capable of detecting acquired and altered DNA, however, only in very closely related strains. If used to compare more distantly related strains (e.g. different species within a genus numerous small changes (i.e. small deletions and point mutations unrelated to the interesting phenotype, would encumber the comparison of 2DBGDs. For this reason a second method, bacterial comparative genomic hybridization (BCGH, was developed to directly compare bacterial genomes to identify gain or loss of genomic DNA. BCGH relies on performing 2DBGD on a pooled sample of genomic DNA from 2 strains to be compared and subsequently hybridizing the resulting 2DBGD blot separately with DNA from each individual strain. Unique spots (hybridization signals represent foreign DNA. The identification of novel DNA is easily achieved by excising the DNA from a dried gel followed by subsequent cloning and sequencing. 2DBGD and BCGH thus represent novel high resolution genome scanning techniques for directly identifying altered and/or acquired DNA.

  2. Algorithms for mapping high-throughput DNA sequences

    DEFF Research Database (Denmark)

    Frellsen, Jes; Menzel, Peter; Krogh, Anders

    2014-01-01

    Abstract High-throughput sequencing (HTS) technologies revolutionized the field of molecular biology by enabling large scale whole genome sequencing as well as a broad range of experiments for studying the cell's inner workings directly on DNA or RNA level. Given the dramatically increased rate...

  3. Perspectives of Integrative Cancer Genomics in Next Generation Sequencing Era

    Directory of Open Access Journals (Sweden)

    So Mee Kwon

    2012-06-01

    Full Text Available The explosive development of genomics technologies including microarrays and next generation sequencing (NGS has provided comprehensive maps of cancer genomes, including the expression of mRNAs and microRNAs, DNA copy numbers, sequence variations, and epigenetic changes. These genome-wide profiles of the genetic aberrations could reveal the candidates for diagnostic and/or prognostic biomarkers as well as mechanistic insights into tumor development and progression. Recent efforts to establish the huge cancer genome compendium and integrative omics analyses, so-called "integromics", have extended our understanding on the cancer genome, showing its daunting complexity and heterogeneity. However, the challenges of the structured integration, sharing, and interpretation of the big omics data still remain to be resolved. Here, we review several issues raised in cancer omics data analysis, including NGS, focusing particularly on the study design and analysis strategies. This might be helpful to understand the current trends and strategies of the rapidly evolving cancer genomics research.

  4. Cancer Genome Sequencing and Its Implications for Personalized Cancer Vaccines

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lijin [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); Goedegebuure, Peter [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Mardis, Elaine R. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); The Genome Institute at Washington University School of Medicine, St. Louis, MO 63108 (United States); Ellis, Matthew J.C. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 (United States); Zhang, Xiuli; Herndon, John M. [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); Fleming, Timothy P. [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Carreno, Beatriz M. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110 (United States); Hansen, Ted H. [The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States); Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Gillanders, William E., E-mail: gillandersw@wudosis.wustl.edu [Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110 (United States); The Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO 63110 (United States)

    2011-11-25

    New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines.

  5. Cancer Genome Sequencing and Its Implications for Personalized Cancer Vaccines

    Directory of Open Access Journals (Sweden)

    William E. Gillanders

    2011-11-01

    Full Text Available New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines.

  6. An integrated semiconductor device enabling non-optical genome sequencing.

    Science.gov (United States)

    Rothberg, Jonathan M; Hinz, Wolfgang; Rearick, Todd M; Schultz, Jonathan; Mileski, William; Davey, Mel; Leamon, John H; Johnson, Kim; Milgrew, Mark J; Edwards, Matthew; Hoon, Jeremy; Simons, Jan F; Marran, David; Myers, Jason W; Davidson, John F; Branting, Annika; Nobile, John R; Puc, Bernard P; Light, David; Clark, Travis A; Huber, Martin; Branciforte, Jeffrey T; Stoner, Isaac B; Cawley, Simon E; Lyons, Michael; Fu, Yutao; Homer, Nils; Sedova, Marina; Miao, Xin; Reed, Brian; Sabina, Jeffrey; Feierstein, Erika; Schorn, Michelle; Alanjary, Mohammad; Dimalanta, Eileen; Dressman, Devin; Kasinskas, Rachel; Sokolsky, Tanya; Fidanza, Jacqueline A; Namsaraev, Eugeni; McKernan, Kevin J; Williams, Alan; Roth, G Thomas; Bustillo, James

    2011-07-20

    The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

  7. Maintenance of Genome Integrity: How Mammalian Cells Orchestrate Genome Duplication by Coordinating Replicative and Specialized DNA Polymerases

    OpenAIRE

    Barnes, Ryan; Eckert, Kristin

    2017-01-01

    Precise duplication of the human genome is challenging due to both its size and sequence complexity. DNA polymerase errors made during replication, repair or recombination are central to creating mutations that drive cancer and aging. Here, we address the regulation of human DNA polymerases, specifically how human cells orchestrate DNA polymerases in the face of stress to complete replication and maintain genome stability. DNA polymerases of the B-family are uniquely adept at accurate genome ...

  8. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd

    Energy Technology Data Exchange (ETDEWEB)

    Fleischmann, R.D.; Adams, M.D.; White, O. [Institute for Genomic Research, Gaithersburg, MD (United States)] [and others

    1995-07-28

    An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism. 46 refs., 4 figs., 4 tabs.

  9. DNA methylation profiling using bisulfite-based epityping of pooled genomic DNA.

    Science.gov (United States)

    Docherty, Sophia J; Davis, Oliver S P; Haworth, Claire M A; Plomin, Robert; Mill, Jonathan

    2010-11-01

    DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however they require large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. DNA pooling approaches are already widely used in large-scale studies of DNA sequence and gene expression. In this paper, we describe the application of this economical DNA pooling technique to the study of DNA methylation profiles. This method generates accurate quantitative assessments of group DNA methylation averages, reducing the time, cost and amount of DNA starting material required for large-scale epigenetic investigation of disease phenotypes.

  10. Detecting long tandem duplications in genomic sequences

    Directory of Open Access Journals (Sweden)

    Audemard Eric

    2012-05-01

    Full Text Available Abstract Background Detecting duplication segments within completely sequenced genomes provides valuable information to address genome evolution and in particular the important question of the emergence of novel functions. The usual approach to gene duplication detection, based on all-pairs protein gene comparisons, provides only a restricted view of duplication. Results In this paper, we introduce ReD Tandem, a software using a flow based chaining algorithm targeted at detecting tandem duplication arrays of moderate to longer length regions, with possibly locally weak similarities, directly at the DNA level. On the A. thaliana genome, using a reference set of tandem duplicated genes built using TAIR,a we show that ReD Tandem is able to predict a large fraction of recently duplicated genes (dS  Conclusions ReD Tandem allows to identify large tandem duplications without any annotation, leading to agnostic identification of tandem duplications. This approach nicely complements the usual protein gene based which ignores duplications involving non coding regions. It is however inherently restricted to relatively recent duplications. By recovering otherwise ignored events, ReD Tandem gives a more comprehensive view of existing evolutionary processes and may also allow to improve existing annotations.

  11. Complete mitochondrial DNA sequences of the threadfin cichlid (Petrochromis trewavasae and the blunthead cichlid (Tropheus moorii and patterns of mitochondrial genome evolution in cichlid fishes.

    Directory of Open Access Journals (Sweden)

    Christoph Fischer

    Full Text Available The cichlid fishes of the East African Great Lakes represent a model especially suited to study adaptive radiation and speciation. With several African cichlid genome projects being in progress, a promising set of closely related genomes is emerging, which is expected to serve as a valuable data base to solve questions on genotype-phenotype relations. The mitochondrial (mt genomes presented here are the first results of the assembly and annotation process for two closely related but eco-morphologically highly distinct Lake Tanganyika cichlids, Petrochromis trewavasae and Tropheus moorii. The genomic sequences comprise 16,588 bp (P. trewavasae and 16,590 bp (T. moorii, and exhibit the typical mitochondrial structure, with 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a non-coding control region. Analyses confirmed that the two species are very closely related with an overall sequence similarity of 96%. We analyzed the newly generated sequences in the phylogenetic context of 21 published labroid fish mitochondrial genomes. Consistent with other vertebrates, the D-loop region was found to evolve faster than protein-coding genes, which in turn are followed by the rRNAs; the tRNAs vary greatly in the rate of sequence evolution, but on average evolve the slowest. Within the group of coding genes, ND6 evolves most rapidly. Codon usage is similar among examined cichlid tribes and labroid families; although a slight shift in usage patterns down the gene tree could be observed. Despite having a clearly different nucleotide composition, ND6 showed a similar codon usage. C-terminal ends of Cox1 exhibit variations, where the varying number of amino acids is related to the structure of the obtained phylogenetic tree. This variation may be of functional relevance for Cox1 synthesis.

  12. A new DNA sequence assembly program.

    Science.gov (United States)

    Bonfield, J K; Smith, K f; Staden, R

    1995-01-01

    We describe the Genome Assembly Program (GAP), a new program for DNA sequence assembly. The program is suitable for large and small projects, a variety of strategies and can handle data from a range of sequencing instruments. It retains the useful components of our previous work, but includes many novel ideas and methods. Many of these methods have been made possible by the program's completely new, and highly interactive, graphical user interface. The program provides many visual clues to the current state of a sequencing project and allows users to interact in intuitive and graphical ways with their data. The program has tools to display and manipulate the various types of data that help to solve and check difficult assemblies, particularly those in repetitive genomes. We have introduced the following new displays: the Contig Selector, the Contig Comparator, the Template Display, the Restriction Enzyme Map and the Stop Codon Map. We have also made it possible to have any number of Contig Editors and Contig Joining Editors running simultaneously even on the same contig. The program also includes a new 'Directed Assembly' algorithm and routines for automatically detecting unfinished segments of sequence, to which it suggests experimental solutions. Images PMID:8559656

  13. Human cellular protein patterns and their link to genome DNA sequence data: usefulness of two-dimensional gel electrophoresis and microsequencing

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Leffers, H;

    1991-01-01

    Analysis of cellular protein patterns by computer-aided 2-dimensional gel electrophoresis together with recent advances in protein sequence analysis have made possible the establishment of comprehensive 2-dimensional gel protein databases that may link protein and DNA information and that offer a...

  14. Qualitatively predicting acetylation and methylation areas in DNA sequences.

    Science.gov (United States)

    Pham, Tho Hoan; Tran, Dang Hung; Ho, Tu Bao; Satou, Kenji; Valiente, Gabriel

    2005-01-01

    Eukaryotic genomes are packaged by the wrapping of DNA around histone octamers to form nucleosomes. Nucleosome occupancy, acetylation, and methylation, which have a major impact on all nuclear processes involving DNA, have been recently mapped across the yeast genome using chromatin immunoprecipitation and DNA microarrays. However, this experimental protocol is laborious and expensive. Moreover, experimental methods often produce noisy results. In this paper, we introduce a computational approach to the qualitative prediction of nucleosome occupancy, acetylation, and methylation areas in DNA sequences. Our method uses support vector machines to discriminate between DNA areas with high and low relative occupancy, acetylation, or methylation, and rank k-gram features based on their support for these DNA modifications. Experimental results on the yeast genome reveal genetic area preferences of nucleosome occupancy, acetylation, and methylation that are consistent with previous studies. Supplementary files are available from http://www.jaist.ac.jp/~tran/nucleosome/.

  15. Genomic Treasure Troves: Complete Genome Sequencing of Herbarium and Insect Museum Specimens

    Science.gov (United States)

    Staats, Martijn; Erkens, Roy H. J.; van de Vossenberg, Bart; Wieringa, Jan J.; Kraaijeveld, Ken; Stielow, Benjamin; Geml, József; Richardson, James E.; Bakker, Freek T.

    2013-01-01

    Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more

  16. Genome Sequence of Lactobacillus rhamnosus ATCC 8530

    OpenAIRE

    Pittet, Vanessa; Ewen, Emily; Bushell, Barry R.; Ziola, Barry

    2012-01-01

    Lactobacillus rhamnosus is found in the human gastrointestinal tract and is important for probiotics. We became interested in L. rhamnosus isolate ATCC 8530 in relation to beer spoilage and hops resistance. We report here the genome sequence of this isolate, along with a brief comparison to other available L. rhamnosus genome sequences.

  17. Repetitive DNA Sequences in Wheat and Its Relatives

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xue-yong; LI Da-yong

    2001-01-01

    Repetitive DNA sequences form a large portion of eukaryote genomes. Using wheat ( Triticum )as a model, the classification, features and functions of repetitive DNA sequences in the Tritieeae grass tribe is reviewed as well as the role of these sequences in genome differentiation, control and regulation of homologous chromosome synapsis and pairing. Transposable elements, as an important portion of dispersed repetitives,may play an essential role in gene mutation of the host. Dynamic models for change of copy number and sequences of the repetitive family are also presented after the models of Charlesworth et al. Application of repetitive DNA sequences in the study of evolution, chromosome fingerprinting and marker assisted gene transfer and breeding are described by taking wheat as an example.

  18. T-DNA Integration Category and Mechanism in Rice Genome

    Institute of Scientific and Technical Information of China (English)

    Jiang WANG; Lin LI; Zhen-Ying SHI; Xin-Shan WAN; Lin-Sheng AN; Jing-Liu ZHANG

    2005-01-01

    T-DNA integration is a key step in the process of plant transformation, which is proven to be important for analyzing T-DNA integration mechanism. The structures of T-DNA right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the T-DNA right border occurred mainly on five nucleotides "TGACA" in inverse repeat (IR)sequence of 25 bp, especially on the third base "A". However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the T-DNA right border, which made the TDNA, carrying vector sequences, integrated into the rice genome. These results seemingly suggest that the IR sequence of the right border plays an important role in the process of T-DNA integration into the rice genome, but is not an essential element. The appearance of vector sequences neighboring the T-DNA right border suggested that before being transferred into the plant cell from Agrobacterium, the entire T-DNA possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the T-DNA right border homologous with plant DNA and filler DNAs were frequently discovered in the integrated position ofT-DNA. Some small regions in the right border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler DNA, through mutual twisting, and then the TDNA was integrated into plant chromosome through a partially homologous recombination mechanism. The appearance of filler DNA would facilitate T-DNA integration. The fragments flanking the T-DNA right border in transformed rice plants could derive from different parts of the inner T-DNA region; that is, disruption and recombination could occur at arbitrary positions in the entire T-DNA, in which the homologous area was comparatively

  19. Human cellular protein patterns and their link to genome DNA sequence data: usefulness of two-dimensional gel electrophoresis and microsequencing

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Leffers, H

    1991-01-01

    Analysis of cellular protein patterns by computer-aided 2-dimensional gel electrophoresis together with recent advances in protein sequence analysis have made possible the establishment of comprehensive 2-dimensional gel protein databases that may link protein and DNA information and that offer...... a global approach to the study of the cell. Using the integrated approach offered by 2-dimensional gel protein databases it is now possible to reveal phenotype specific protein (or proteins), to microsequence them, to search for homology with previously identified proteins, to clone the cDNAs, to assign...... partial protein sequence to genes for which the full DNA sequence and the chromosome location is known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Human 2-dimensional gel protein databases are becoming...

  20. DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

    Directory of Open Access Journals (Sweden)

    Cheng-Yao eChen

    2014-06-01

    Full Text Available Next-generation sequencing (NGS technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

  1. Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

    Directory of Open Access Journals (Sweden)

    Luo Ming-Cheng

    2011-01-01

    Full Text Available Abstract Background Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence. Results An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in Aegilops tauschii-the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of Ae. tauschii accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of Ae. tauschii accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire Ae. tauschii genome. To assess the false positive SNP discovery rate, DNA

  2. Human Genome Sequencing in Health and Disease

    Science.gov (United States)

    Gonzaga-Jauregui, Claudia; Lupski, James R.; Gibbs, Richard A.

    2013-01-01

    Following the “finished,” euchromatic, haploid human reference genome sequence, the rapid development of novel, faster, and cheaper sequencing technologies is making possible the era of personalized human genomics. Personal diploid human genome sequences have been generated, and each has contributed to our better understanding of variation in the human genome. We have consequently begun to appreciate the vastness of individual genetic variation from single nucleotide to structural variants. Translation of genome-scale variation into medically useful information is, however, in its infancy. This review summarizes the initial steps undertaken in clinical implementation of personal genome information, and describes the application of whole-genome and exome sequencing to identify the cause of genetic diseases and to suggest adjuvant therapies. Better analysis tools and a deeper understanding of the biology of our genome are necessary in order to decipher, interpret, and optimize clinical utility of what the variation in the human genome can teach us. Personal genome sequencing may eventually become an instrument of common medical practice, providing information that assists in the formulation of a differential diagnosis. We outline herein some of the remaining challenges. PMID:22248320

  3. Genome size evolution in pufferfish: an insight from BAC clone-based Diodon holocanthus genome sequencing

    Directory of Open Access Journals (Sweden)

    Gan Xiaoni

    2010-06-01

    Full Text Available Abstract Background Variations in genome size within and between species have been observed since the 1950 s in diverse taxonomic groups. Serving as model organisms, smooth pufferfish possess the smallest vertebrate genomes. Interestingly, spiny pufferfish from its sister family have genome twice as large as smooth pufferfish. Therefore, comparative genomic analysis between smooth pufferfish and spiny pufferfish is useful for our understanding of genome size evolution in pufferfish. Results Ten BAC clones of a spiny pufferfish Diodon holocanthus were randomly selected and shotgun sequenced. In total, 776 kb of non-redundant sequences without gap representing 0.1% of the D. holocanthus genome were identified, and 77 distinct genes were predicted. In the sequenced D. holocanthus genome, 364 kb is homologous with 265 kb of the Takifugu rubripes genome, and 223 kb is homologous with 148 kb of the Tetraodon nigroviridis genome. The repetitive DNA accounts for 8% of the sequenced D. holocanthus genome, which is higher than that in the T. rubripes genome (6.89% and that in the Te. nigroviridis genome (4.66%. In the repetitive DNA, 76% is retroelements which account for 6% of the sequenced D. holocanthus genome and belong to known families of transposable elements. More than half of retroelements were distributed within genes. In the non-homologous regions, repeat element proportion in D. holocanthus genome increased to 10.6% compared with T. rubripes and increased to 9.19% compared with Te. nigroviridis. A comparison of 10 well-defined orthologous genes showed that the average intron size (566 bp in D. holocanthus genome is significantly longer than that in the smooth pufferfish genome (435 bp. Conclusion Compared with the smooth pufferfish, D. holocanthus has a low gene density and repeat elements rich genome. Genome size variation between D. holocanthus and the smooth pufferfish exhibits as length variation between homologous region and different

  4. Genome-wide mapping of DNA strand breaks.

    Directory of Open Access Journals (Sweden)

    Frédéric Leduc

    Full Text Available Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP, uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

  5. The genome sequence of parrot bornavirus 5.

    Science.gov (United States)

    Guo, Jianhua; Tizard, Ian

    2015-12-01

    Although several new avian bornaviruses have recently been described, information on their evolution, virulence, and sequence are often limited. Here we report the complete genome sequence of parrot bornavirus 5 (PaBV-5) isolated from a case of proventricular dilatation disease in a Palm cockatoo (Probosciger aterrimus). The complete genome consists of 8842 nucleotides with distinct 5' and 3' end sequences. This virus shares nucleotide sequence identities of 69-74 % with other bornaviruses in the genomic regions excluding the 5' and 3' terminal sequences. Phylogenetic analysis based on the genomic regions demonstrated this new isolate is an isolated branch within the clade that includes the aquatic bird bornaviruses and the passerine bornaviruses. Based on phylogenetic analyses and its low nucleotide sequence identities with other bornavirus, we support the proposal that PaBV-5 be assigned to a new bornavirus species:- Psittaciform 2 bornavirus.

  6. Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner

    DEFF Research Database (Denmark)

    Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan

    2009-01-01

    MOTIVATION: The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary......' simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners...... heuristics. RESULTS: We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created 'perfect...

  7. An efficient procedure for plant organellar genome assembly, based on whole genome data from the 454 GS FLX sequencing platform

    Directory of Open Access Journals (Sweden)

    Zhang Tongwu

    2011-11-01

    Full Text Available Abstract Motivation Complete organellar genome sequences (chloroplasts and mitochondria provide valuable resources and information for studying plant molecular ecology and evolution. As high-throughput sequencing technology advances, it becomes the norm that a shotgun approach is used to obtain complete genome sequences. Therefore, to assemble organellar sequences from the whole genome, shotgun reads are inevitable. However, associated techniques are often cumbersome, time-consuming, and difficult, because true organellar DNA is difficult to separate efficiently from nuclear copies, which have been transferred to the nucleus through the course of evolution. Results We report a new, rapid procedure for plant chloroplast and mitochondrial genome sequencing and assembly using the Roche/454 GS FLX platform. Plant cells can contain multiple copies of the organellar genomes, and there is a significant correlation between the depth of sequence reads in contigs and the number of copies of the genome. Without isolating organellar DNA from the mixture of nuclear and organellar DNA for sequencing, we retrospectively extracted assembled contigs of either chloroplast or mitochondrial sequences from the whole genome shotgun data. Moreover, the contig connection graph property of Newbler (a platform-specific sequence assembler ensures an efficient final assembly. Using this procedure, we assembled both chloroplast and mitochondrial genomes of a resurrection plant, Boea hygrometrica, with high fidelity. We also present information and a minimal sequence dataset as a reference for the assembly of other plant organellar genomes.

  8. The diploid genome sequence of an individual human.

    Directory of Open Access Journals (Sweden)

    Samuel Levy

    2007-09-01

    Full Text Available Presented here is a genome sequence of an individual human. It was produced from approximately 32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel included 3,213,401 single nucleotide polymorphisms (SNPs, 53,823 block substitutions (2-206 bp, 292,102 heterozygous insertion/deletion events (indels(1-571 bp, 559,473 homozygous indels (1-82,711 bp, 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.

  9. Complete Genome Sequence of Canine Papillomavirus Type 16

    OpenAIRE

    Luff, Jennifer; Mader, Michelle; Britton, Monica; Fass, Joseph; Rowland, Peter; Orr, Carolyn; Schlegel, Richard; Yuan, Hang

    2015-01-01

    Papillomaviruses are epitheliotropic, circular, double-stranded DNA viruses within the family Papillomaviridae that are associated with benign and malignant tumors in humans and animals. We report the complete genome sequence of canine papillomavirus type 16 identified within multiple pigmented cutaneous plaques and squamous cell carcinoma from an intact female Basenji dog.

  10. Intra-species sequence comparisons for annotating genomes

    Energy Technology Data Exchange (ETDEWEB)

    Boffelli, Dario; Weer, Claire V.; Weng, Li; Lewis, Keith D.; Shoukry, Malak I.; Pachter, Lior; Keys, David N.; Rubin, Edward M.

    2004-07-15

    Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intra-species sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents and a set of genomic intervals amplified, resequenced and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom and raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species. The sequence data from this study has been submitted to GenBank under accession nos. AY667278-AY667407.

  11. Reduced representation sequencing: a success in maize and a promise for other plant genomes.

    Science.gov (United States)

    Barbazuk, W Brad; Bedell, Joseph A; Rabinowicz, Pablo D

    2005-08-01

    Plant, and particularly cereal genomes, are challenging to sequence due to their large size and high repetitive DNA content. Gene-enrichment strategies are alternative or complementary approaches to complete genome sequencing that yield, rapidly and inexpensively, useful sequence data from large and complex genomes. The maize genome is large (2.7 Gbp) and contains large amounts of conserved repetitive elements. Furthermore, the high allelic diversity found between maize inbred lines may necessitate sequencing several inbred lines in order to recover the maize "gene pool". Two gene-enrichment approaches, methylation filtration (MF) and high C(o)t (HC) sequencing have been tested in maize and their ability to sample the gene space has been examined. Combined with other genomic sequencing strategies, gene-enriched genomic sequencing is a practical way to examine the maize gene pool, to order and orient the genic sequences on the genome, and to enable investigation of gene content of other complex plant genomes.

  12. Real-time, portable genome sequencing for Ebola surveillance

    Science.gov (United States)

    Bore, Joseph Akoi; Koundouno, Raymond; Dudas, Gytis; Mikhail, Amy; Ouédraogo, Nobila; Afrough, Babak; Bah, Amadou; Baum, Jonathan HJ; Becker-Ziaja, Beate; Boettcher, Jan-Peter; Cabeza-Cabrerizo, Mar; Camino-Sanchez, Alvaro; Carter, Lisa L.; Doerrbecker, Juiliane; Enkirch, Theresa; Dorival, Isabel Graciela García; Hetzelt, Nicole; Hinzmann, Julia; Holm, Tobias; Kafetzopoulou, Liana Eleni; Koropogui, Michel; Kosgey, Abigail; Kuisma, Eeva; Logue, Christopher H; Mazzarelli, Antonio; Meisel, Sarah; Mertens, Marc; Michel, Janine; Ngabo, Didier; Nitzsche, Katja; Pallash, Elisa; Patrono, Livia Victoria; Portmann, Jasmine; Repits, Johanna Gabriella; Rickett, Natasha Yasmin; Sachse, Andrea; Singethan, Katrin; Vitoriano, Inês; Yemanaberhan, Rahel L; Zekeng, Elsa G; Trina, Racine; Bello, Alexander; Sall, Amadou Alpha; Faye, Ousmane; Faye, Oumar; Magassouba, N’Faly; Williams, Cecelia V.; Amburgey, Victoria; Winona, Linda; Davis, Emily; Gerlach, Jon; Washington, Franck; Monteil, Vanessa; Jourdain, Marine; Bererd, Marion; Camara, Alimou; Somlare, Hermann; Camara, Abdoulaye; Gerard, Marianne; Bado, Guillaume; Baillet, Bernard; Delaune, Déborah; Nebie, Koumpingnin Yacouba; Diarra, Abdoulaye; Savane, Yacouba; Pallawo, Raymond Bernard; Gutierrez, Giovanna Jaramillo; Milhano, Natacha; Roger, Isabelle; Williams, Christopher J; Yattara, Facinet; Lewandowski, Kuiama; Taylor, Jamie; Rachwal, Philip; Turner, Daniel; Pollakis, Georgios; Hiscox, Julian A.; Matthews, David A.; O’Shea, Matthew K.; Johnston, Andrew McD; Wilson, Duncan; Hutley, Emma; Smit, Erasmus; Di Caro, Antonino; Woelfel, Roman; Stoecker, Kilian; Fleischmann, Erna; Gabriel, Martin; Weller, Simon A.; Koivogui, Lamine; Diallo, Boubacar; Keita, Sakoba; Rambaut, Andrew; Formenty, Pierre; Gunther, Stephan; Carroll, Miles W.

    2016-01-01

    The Ebola virus disease (EVD) epidemic in West Africa is the largest on record, responsible for >28,599 cases and >11,299 deaths 1. Genome sequencing in viral outbreaks is desirable in order to characterize the infectious agent to determine its evolutionary rate, signatures of host adaptation, identification and monitoring of diagnostic targets and responses to vaccines and treatments. The Ebola virus genome (EBOV) substitution rate in the Makona strain has been estimated at between 0.87 × 10−3 to 1.42 × 10−3 mutations per site per year. This is equivalent to 16 to 27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic 2-7. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought-after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions 8. Genomic surveillance during the epidemic has been sporadic due to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities 9. In order to address this problem, we devised a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. Here we present sequence data and analysis of 142 Ebola virus (EBOV) samples collected during the period March to October 2015. We were able to generate results in less than 24 hours after receiving an Ebola positive sample, with the sequencing process taking as little as 15-60 minutes. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks. PMID:26840485

  13. Draft genome sequence of the silver pomfret fish, Pampus argenteus.

    Science.gov (United States)

    AlMomin, Sabah; Kumar, Vinod; Al-Amad, Sami; Al-Hussaini, Mohsen; Dashti, Talal; Al-Enezi, Khaznah; Akbar, Abrar

    2016-01-01

    Silver pomfret, Pampus argenteus, is a fish species from coastal waters. Despite its high commercial value, this edible fish has not been sequenced. Hence, its genetic and genomic studies have been limited. We report the first draft genome sequence of the silver pomfret obtained using a Next Generation Sequencing (NGS) technology. We assembled 38.7 Gb of nucleotides into scaffolds of 350 Mb with N50 of about 1.5 kb, using high quality paired end reads. These scaffolds represent 63.7% of the estimated silver pomfret genome length. The newly sequenced and assembled genome has 11.06% repetitive DNA regions, and this percentage is comparable to that of the tilapia genome. The genome analysis predicted 16 322 genes. About 91% of these genes showed homology with known proteins. Many gene clusters were annotated to protein and fatty-acid metabolism pathways that may be important in the context of the meat texture and immune system developmental processes. The reference genome can pave the way for the identification of many other genomic features that could improve breeding and population-management strategies, and it can also help characterize the genetic diversity of P. argenteus.

  14. The complete chloroplast genome sequence of Abies nephrolepis (Pinaceae: Abietoideae

    Directory of Open Access Journals (Sweden)

    Dong-Keun Yi

    2016-06-01

    Full Text Available The plant chloroplast (cp genome has maintained a relatively conserved structure and gene content throughout evolution. Cp genome sequences have been used widely for resolving evolutionary and phylogenetic issues at various taxonomic levels of plants. Here, we report the complete cp genome of Abies nephrolepis. The A. nephrolepis cp genome is 121,336 base pairs (bp in length including a pair of short inverted repeat regions (IRa and IRb of 139 bp each separated by a small single copy (SSC region of 54,323 bp (SSC and a large single copy region of 66,735 bp (LSC. It contains 114 genes, 68 of which are protein coding genes, 35 tRNA and four rRNA genes, six open reading frames, and one pseudogene. Seventeen repeat units and 64 simple sequence repeats (SSR have been detected in A. nephrolepis cp genome. Large IR sequences locate in 42-kb inversion points (1186 bp. The A. nephrolepis cp genome is identical to Abies koreana’s which is closely related to taxa. Pairwise comparison between two cp genomes revealed 140 polymorphic sites in each. Complete cp genome sequence of A. nephrolepis has a significant potential to provide information on the evolutionary pattern of Abietoideae and valuable data for development of DNA markers for easy identification and classification.

  15. Hellbender genome sequences shed light on genomic expansion at the base of crown salamanders.

    Science.gov (United States)

    Sun, Cheng; Mueller, Rachel Lockridge

    2014-07-01

    Among animals, genome sizes range from 20 Mb to 130 Gb, with 380-fold variation across vertebrates. Most of the largest vertebrate genomes are found in salamanders, an amphibian clade of 660 species. Thus, salamanders are an important system for studying causes and consequences of genomic gigantism. Previously, we showed that plethodontid salamander genomes accumulate higher levels of long terminal repeat (LTR) retrotransposons than do other vertebrates, although the evolutionary origins of such sequences remained unexplored. We also showed that some salamanders in the family Plethodontidae have relatively slow rates of DNA loss through small insertions and deletions. Here, we present new data from Cryptobranchus alleganiensis, the hellbender. Cryptobranchus and Plethodontidae span the basal phylogenetic split within salamanders; thus, analyses incorporating these taxa can shed light on the genome of the ancestral crown salamander lineage, which underwent expansion. We show that high levels of LTR retrotransposons likely characterize all crown salamanders, suggesting that disproportionate expansion of this transposable element (TE) class contributed to genomic expansion. Phylogenetic and age distribution analyses of salamander LTR retrotransposons indicate that salamanders' high TE levels reflect persistence and diversification of ancestral TEs rather than horizontal transfer events. Finally, we show that relatively slow DNA loss rates through small indels likely characterize all crown salamanders, suggesting that a decreased DNA loss rate contributed to genomic expansion at the clade's base. Our identification of shared genomic features across phylogenetically distant salamanders is a first step toward identifying the evolutionary processes underlying accumulation and persistence of high levels of repetitive sequence in salamander genomes.

  16. Comprehensive DNA methylation analysis of the Aedes aegypti genome

    Science.gov (United States)

    Falckenhayn, Cassandra; Carneiro, Vitor Coutinho; de Mendonça Amarante, Anderson; Schmid, Katharina; Hanna, Katharina; Kang, Seokyoung; Helm, Mark; Dimopoulos, George; Fantappié, Marcelo Rosado; Lyko, Frank

    2016-01-01

    Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation. PMID:27805064

  17. Genomic selective constraints in murid noncoding DNA.

    Directory of Open Access Journals (Sweden)

    Daniel J Gaffney

    2006-11-01

    Full Text Available Recent work has suggested that there are many more selectively constrained, functional noncoding than coding sites in mammalian genomes. However, little is known about how selective constraint varies amongst different classes of noncoding DNA. We estimated the magnitude of selective constraint on a large dataset of mouse-rat gene orthologs and their surrounding noncoding DNA. Our analysis indicates that there are more than three times as many selectively constrained, nonrepetitive sites within noncoding DNA as in coding DNA in murids. The majority of these constrained noncoding sites appear to be located within intergenic regions, at distances greater than 5 kilobases from known genes. Our study also shows that in murids, intron length and mean intronic selective constraint are negatively correlated with intron ordinal number. Our results therefore suggest that functional intronic sites tend to accumulate toward the 5' end of murid genes. Our analysis also reveals that mean number of selectively constrained noncoding sites varies substantially with the function of the adjacent gene. We find that, among others, developmental and neuronal genes are associated with the greatest numbers of putatively functional noncoding sites compared with genes involved in electron transport and a variety of metabolic processes. Combining our estimates of the total number of constrained coding and noncoding bases we calculate that over twice as many deleterious mutations have occurred in intergenic regions as in known genic sequence and that the total genomic deleterious point mutation rate is 0.91 per diploid genome, per generation. This estimated rate is over twice as large as a previous estimate in murids.

  18. The Release 6 reference sequence of the Drosophila melanogaster genome

    Science.gov (United States)

    Carlson, Joseph W.; Wan, Kenneth H.; Park, Soo; Mendez, Ivonne; Galle, Samuel E.; Booth, Benjamin W.; Pfeiffer, Barret D.; George, Reed A.; Svirskas, Robert; Krzywinski, Martin; Schein, Jacqueline; Accardo, Maria Carmela; Damia, Elisabetta; Messina, Giovanni; Méndez-Lago, María; de Pablos, Beatriz; Demakova, Olga V.; Andreyeva, Evgeniya N.; Boldyreva, Lidiya V.; Marra, Marco; Carvalho, A. Bernardo; Dimitri, Patrizio; Villasante, Alfredo; Zhimulev, Igor F.; Rubin, Gerald M.; Karpen, Gary H.

    2015-01-01

    Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads. PMID:25589440

  19. DNA Sequencing in Cultural Heritage.

    Science.gov (United States)

    Vai, Stefania; Lari, Martina; Caramelli, David

    2016-02-01

    During the last three decades, DNA analysis on degraded samples revealed itself as an important research tool in anthropology, archaeozoology, molecular evolution, and population genetics. Application on topics such as determination of species origin of prehistoric and historic objects, individual identification of famous personalities, characterization of particular samples important for historical, archeological, or evolutionary reconstructions, confers to the paleogenetics an important role also for the enhancement of cultural heritage. A really fast improvement in methodologies in recent years led to a revolution that permitted recovering even complete genomes from highly degraded samples with the possibility to go back in time 400,000 years for samples from temperate regions and 700,000 years for permafrozen remains and to analyze even more recent material that has been subjected to hard biochemical treatments. Here we propose a review on the different methodological approaches used so far for the molecular analysis of degraded samples and their application on some case studies.

  20. Assessment of whole genome amplification for sequence capture and massively parallel sequencing.

    Directory of Open Access Journals (Sweden)

    Johanna Hasmats

    Full Text Available Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74% of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.

  1. Assessment of whole genome amplification for sequence capture and massively parallel sequencing.

    Science.gov (United States)

    Hasmats, Johanna; Gréen, Henrik; Orear, Cedric; Validire, Pierre; Huss, Mikael; Käller, Max; Lundeberg, Joakim

    2014-01-01

    Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.

  2. DNA Shape Dominates Sequence Affinity in Nucleosome Formation

    Science.gov (United States)

    Freeman, Gordon S.; Lequieu, Joshua P.; Hinckley, Daniel M.; Whitmer, Jonathan K.; de Pablo, Juan J.

    2014-10-01

    Nucleosomes provide the basic unit of compaction in eukaryotic genomes, and the mechanisms that dictate their position at specific locations along a DNA sequence are of central importance to genetics. In this Letter, we employ molecular models of DNA and proteins to elucidate various aspects of nucleosome positioning. In particular, we show how DNA's histone affinity is encoded in its sequence-dependent shape, including subtle deviations from the ideal straight B-DNA form and local variations of minor groove width. By relying on high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome positioning.

  3. Sequencing Crop Genomes: A Gateway to Improve Tropical Agriculture.

    Science.gov (United States)

    Thottathil, Gincy Paily; Jayasekaran, Kandakumar; Othman, Ahmad Sofiman

    2016-02-01

    Agricultural development in the tropics lags behind development in the temperate latitudes due to the lack of advanced technology, and various biotic and abiotic factors. To cope with the increasing demand for food and other plant-based products, improved crop varieties have to be developed. To breed improved varieties, a better understanding of crop genetics is necessary. With the advent of next-generation DNA sequencing technologies, many important crop genomes have been sequenced. Primary importance has been given to food crops, including cereals, tuber crops, vegetables, and fruits. The DNA sequence information is extremely valuable for identifying key genes controlling important agronomic traits and for identifying genetic variability among the cultivars. However, massive DNA re-sequencing and gene expression studies have to be performed to substantially improve our understanding of crop genetics. Application of the knowledge obtained from the genomes, transcriptomes, expression studies, and epigenetic studies would enable the development of improved varieties and may lead to a second green revolution. The applications of next generation DNA sequencing technologies in crop improvement, its limitations, future prospects, and the features of important crop genome projects are reviewed herein.

  4. Strategies for complete plastid genome sequencing.

    Science.gov (United States)

    Twyford, Alex D; Ness, Rob W

    2016-10-28

    Plastid sequencing is an essential tool in the study of plant evolution. This high-copy organelle is one of the most technically accessible regions of the genome, and its sequence conservation makes it a valuable region for comparative genome evolution, phylogenetic analysis and population studies. Here, we discuss recent innovations and approaches for de novo plastid assembly that harness genomic tools. We focus on technical developments including low-cost sequence library preparation approaches for genome skimming, enrichment via hybrid baits and methylation-sensitive capture, sequence platforms with higher read outputs and longer read lengths, and automated tools for assembly. These developments allow for a much more streamlined assembly than via conventional short-range PCR. Although newer methods make complete plastid sequencing possible for any land plant or green alga, there are still challenges for producing finished plastomes particularly from herbarium material or from structurally divergent plastids such as those of parasitic plants.

  5. Volume visualization of multiple alignment of genomic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Shah, Nameeta; Weber, Gunther H.; Dillard, Scott E.; Hamann, Bernd

    2004-05-01

    Genomes of hundreds of species have been sequenced to date and many more are being sequenced. As more and more sequence data sets become available, and as the challenge of comparing these massive ''billion basepair DNA sequences'' becomes substantial, so does the need for more powerful tools supporting the exploration of these data sets. Similarity score data used to compare aligned DNA sequences is inherently one-dimensional. One-dimensional (1D) representations of these data sets do not effectively utilize screen real estate. We present a technique to arrange 1D data in 3D space to allow us to apply state-of-the-art interactive volume visualization techniques for data exploration. We provide results for aligned DNA sequence data and compare it with traditional 1D line plots. Our technique, coupled with 1D line plots, results in effective multiresolution visualization of very large aligned sequence data sets.

  6. Building a model: developing genomic resources for common milkweed (Asclepias syriaca with low coverage genome sequencing

    Directory of Open Access Journals (Sweden)

    Weitemier Kevin

    2011-05-01

    Full Text Available Abstract Background Milkweeds (Asclepias L. have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L. could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp and 5S rDNA (120 bp sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp, with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae unigenes (median coverage of 0.29× and 66% of single copy orthologs (COSII in asterids (median coverage of 0.14×. From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites and phylogenetics (low-copy nuclear genes studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species

  7. Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

    Science.gov (United States)

    Straub, Shannon C K; Fishbein, Mark; Livshultz, Tatyana; Foster, Zachary; Parks, Matthew; Weitemier, Kevin; Cronn, Richard C; Liston, Aaron

    2011-05-04

    Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first

  8. Plantagora: modeling whole genome sequencing and assembly of plant genomes.

    Directory of Open Access Journals (Sweden)

    Roger Barthelson

    Full Text Available BACKGROUND: Genomics studies are being revolutionized by the next generation sequencing technologies, which have made whole genome sequencing much more accessible to the average researcher. Whole genome sequencing with the new technologies is a developing art that, despite the large volumes of data that can be produced, may still fail to provide a clear and thorough map of a genome. The Plantagora project was conceived to address specifically the gap between having the technical tools for genome sequencing and knowing precisely the best way to use them. METHODOLOGY/PRINCIPAL FINDINGS: For Plantagora, a platform was created for generating simulated reads from several different plant genomes of different sizes. The resulting read files mimicked either 454 or Illumina reads, with varying paired end spacing. Thousands of datasets of reads were created, most derived from our primary model genome, rice chromosome one. All reads were assembled with different software assemblers, including Newbler, Abyss, and SOAPdenovo, and the resulting assemblies were evaluated by an extensive battery of metrics chosen for these studies. The metrics included both statistics of the assembly sequences and fidelity-related measures derived by alignment of the assemblies to the original genome source for the reads. The results were presented in a website, which includes a data graphing tool, all created to help the user compare rapidly the feasibility and effectiveness of different sequencing and assembly strategies prior to testing an approach in the lab. Some of our own conclusions regarding the different strategies were also recorded on the website. CONCLUSIONS/SIGNIFICANCE: Plantagora provides a substantial body of information for comparing different approaches to sequencing a plant genome, and some conclusions regarding some of the specific approaches. Plantagora also provides a platform of metrics and tools for studying the process of sequencing and assembly

  9. Monitoring Genomic Sequences during SELEX Using High-Throughput Sequencing: Neutral SELEX

    Science.gov (United States)

    Chen, Doris; Lorenz, Christina; Schroeder, Renée

    2010-01-01

    Background SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection. Methodology/Principal Findings To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection. Conclusions/Significance Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers. PMID:20161784

  10. Monitoring genomic sequences during SELEX using high-throughput sequencing: neutral SELEX.

    Directory of Open Access Journals (Sweden)

    Bob Zimmermann

    Full Text Available BACKGROUND: SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection. CONCLUSIONS/SIGNIFICANCE: Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.

  11. Whole genome and transcriptome sequencing of a B3 thymoma.

    Directory of Open Access Journals (Sweden)

    Iacopo Petrini

    Full Text Available Molecular pathology of thymomas is poorly understood. Genomic aberrations are frequently identified in tumors but no extensive sequencing has been reported in thymomas. Here we present the first comprehensive view of a B3 thymoma at whole genome and transcriptome levels. A 55-year-old Caucasian female underwent complete resection of a stage IVA B3 thymoma. RNA and DNA were extracted from a snap frozen tumor sample with a fraction of cancer cells over 80%. We performed array comparative genomic hybridization using Agilent platform, transcriptome sequencing using HiSeq 2000 (Illumina and whole genome sequencing using Complete Genomics Inc platform. Whole genome sequencing determined, in tumor and normal, the sequence of both alleles in more than 95% of the reference genome (NCBI Build 37. Copy number (CN aberrations were comparable with those previously described for B3 thymomas, with CN gain of chromosome 1q, 5, 7 and X and CN loss of 3p, 6, 11q42.2-qter and q13. One translocation t(11;X was identified by whole genome sequencing and confirmed by PCR and Sanger sequencing. Ten single nucleotide variations (SNVs and 2 insertion/deletions (INDELs were identified; these mutations resulted in non-synonymous amino acid changes or affected splicing sites. The lack of common cancer-associated mutations in this patient suggests that thymomas may evolve through mechanisms distinctive from other tumor types, and supports the rationale for additional high-throughput sequencing screens to better understand the somatic genetic architecture of thymoma.

  12. Artificial duplicate reads in sequencing data of 454 Genome Sequencer FLX System

    Institute of Scientific and Technical Information of China (English)

    Hui Dong; Yangyi Chen; Yan Shen; Shengyue Wang; Guoping Zhao; Weirong Jin

    2011-01-01

    The 454 Genome Sequencer (GS) FLX System is one of the next-generation sequencing systems featured by long reads, high accuracy, and ultra-high throughput.Based on the mechanism of emulsion PCR, a unique DNA template would only generate a unique sequence read after being amplified and sequenced on GS FLX.However,biased amplification of DNA templates might occur in the process of emulsion PCR, which results in production of artificial duplicate reads.Under the condition that each DNA template is unique to another, 3.49%-18.14% of total reads in GS FLX-sequencing data were found to be artificial duplicate reads.These duplicate reads may lead to misunderstanding of sequencing data and special attention should be paid to the potential biases they introduced to the data.

  13. A DNA minor groove electronegative potential genome map based on photo-chemical probing

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter Eigil; Hansen, Morten

    2011-01-01

    The double-stranded DNA of the genome contains both sequence information directly relating to the protein and RNA coding as well as functional and structural information relating to protein recognition. Only recently is the importance of DNA shape in this recognition process being fully appreciated...... resolution of any genome, and it is illustrated how such detailed studies of this sequence dependent, inherent property of the DNA may reflect on genome organization, gene expression and chromosomal condensation....

  14. Current-voltage characteristics of double-strand DNA sequences

    Science.gov (United States)

    Bezerril, L. M.; Moreira, D. A.; Albuquerque, E. L.; Fulco, U. L.; de Oliveira, E. L.; de Sousa, J. S.

    2009-09-01

    We use a tight-binding formulation to investigate the transmissivity and the current-voltage (I-V) characteristics of sequences of double-strand DNA molecules. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of artificial sequences (the long-range correlated Fibonacci and Rudin-Shapiro one) and a random sequence, which is a kind of prototype of a short-range correlated system. The random sequence is presented here with the same first neighbors pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the transmissivity spectra, although the I-V curves seem to be mostly influenced by the short-range correlations.

  15. Current-voltage characteristics of double-strand DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Bezerril, L.M.; Moreira, D.A. [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Albuquerque, E.L., E-mail: eudenilson@dfte.ufrn.b [Departamento de Fisica, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Fulco, U.L. [Departamento de Biofisica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970, Natal-RN (Brazil); Oliveira, E.L. de; Sousa, J.S. de [Departamento de Fisica, Universidade Federal do Ceara, 60455-760, Fortaleza-CE (Brazil)

    2009-09-07

    We use a tight-binding formulation to investigate the transmissivity and the current-voltage (I-V) characteristics of sequences of double-strand DNA molecules. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of artificial sequences (the long-range correlated Fibonacci and Rudin-Shapiro one) and a random sequence, which is a kind of prototype of a short-range correlated system. The random sequence is presented here with the same first neighbors pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the transmissivity spectra, although the I-V curves seem to be mostly influenced by the short-range correlations.

  16. Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation* ♦

    Science.gov (United States)

    Chakraborty, Anirban; Wakamiya, Maki; Venkova-Canova, Tatiana; Pandita, Raj K.; Aguilera-Aguirre, Leopoldo; Sarker, Altaf H.; Singh, Dharmendra Kumar; Hosoki, Koa; Wood, Thomas G.; Sharma, Gulshan; Cardenas, Victor; Sarkar, Partha S.; Sur, Sanjiv; Pandita, Tej K.; Boldogh, Istvan; Hazra, Tapas K.

    2015-01-01

    Why mammalian cells possess multiple DNA glycosylases (DGs) with overlapping substrate ranges for repairing oxidatively damaged bases via the base excision repair (BER) pathway is a long-standing question. To determine the biological role of these DGs, null animal models have been generated. Here, we report the generation and characterization of mice lacking Neil2 (Nei-like 2). As in mice deficient in each of the other four oxidized base-specific DGs (OGG1, NTH1, NEIL1, and NEIL3), Neil2-null mice show no overt phenotype. However, middle-aged to old Neil2-null mice show the accumulation of oxidative genomic damage, mostly in the transcribed regions. Immuno-pulldown analysis from wild-type (WT) mouse tissue showed the association of NEIL2 with RNA polymerase II, along with Cockayne syndrome group B protein, TFIIH, and other BER proteins. Chromatin immunoprecipitation analysis from mouse tissue showed co-occupancy of NEIL2 and RNA polymerase II only on the transcribed genes, consistent with our earlier in vitro findings on NEIL2's role in transcription-coupled BER. This study provides the first in vivo evidence of genomic region-specific repair in mammals. Furthermore, telomere loss and genomic instability were observed at a higher frequency in embryonic fibroblasts from Neil2-null mice than from the WT. Moreover, Neil2-null mice are much more responsive to inflammatory agents than WT mice. Taken together, our results underscore the importance of NEIL2 in protecting mammals from the development of various pathologies that are linked to genomic instability and/or inflammation. NEIL2 is thus likely to play an important role in long term genomic maintenance, particularly in long-lived mammals such as humans. PMID:26245904

  17. Sequence Determination from Overlapping Fragments: A Simple Model of Whole-Genome Shotgun Sequencing

    Science.gov (United States)

    Derrida, Bernard; Fink, Thomas M.

    2002-02-01

    Assembling fragments randomly sampled from along a sequence is the basis of whole-genome shotgun sequencing, a technique used to map the DNA of the human and other genomes. We calculate the probability that a random sequence can be recovered from a collection of overlapping fragments. We provide an exact solution for an infinite alphabet and in the case of constant overlaps. For the general problem we apply two assembly strategies and give the probability that the assembly puzzle can be solved in the limit of infinitely many fragments.

  18. Extraction of high-quality genomic DNA from Ectocarpus.

    Science.gov (United States)

    Coelho, Susana M; Scornet, Delphine; Rousvoal, Sylvie; Peters, Nick; Dartevelle, Laurence; Peters, Akira F; Cock, J Mark

    2012-03-01

    For some applications, such as genome sequencing and high-throughput genotyping with multiple markers, it is necessary to use high-quality genomic DNA. This article describes how to obtain several micrograms of high-quality, cesium chloride-purified DNA from 1 g of Ectocarpus filaments. We also recommend using DNA of this quality for quantitative RT-PCR control reactions. However, simpler, more rapid, kit-based methods are preferable for experiments that involve the treatment of large numbers of individuals, such as genotyping large populations with a small number of markers or PCR screening of large populations.

  19. Next-Generation Sequencing and Genome Editing in Plant Virology

    OpenAIRE

    Hadidi, Ahmed; Flores, Ricardo; Candresse, Thierry; Barba, Marina

    2016-01-01

    Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant vir...

  20. Microbial species delineation using whole genome sequences

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Mukherjee, Supratim; Ivanova, Natalia; Mavrommatics, Kostas; Pati, Amrita; Konstantinidis, Konstantinos

    2014-10-20

    Species assignments in prokaryotes use a manual, poly-phasic approach utilizing both phenotypic traits and sequence information of phylogenetic marker genes. With thousands of genomes being sequenced every year, an automated, uniform and scalable approach exploiting the rich genomic information in whole genome sequences is desired, at least for the initial assignment of species to an organism. We have evaluated pairwise genome-wide Average Nucleotide Identity (gANI) values and alignment fractions (AFs) for nearly 13,000 genomes using our fast implementation of the computation, identifying robust and widely applicable hard cut-offs for species assignments based on AF and gANI. Using these cutoffs, we generated stable species-level clusters of organisms, which enabled the identification of several species mis-assignments and facilitated the assignment of species for organisms without species definitions.

  1. Genomic Prediction from Whole Genome Sequence in Livestock: The 1000 Bull Genomes Project

    DEFF Research Database (Denmark)

    Hayes, Benjamin J; MacLeod, Iona M; Daetwyler, Hans D

    Advantages of using whole genome sequence data to predict genomic estimated breeding values (GEBV) include better persistence of accuracy of GEBV across generations and more accurate GEBV across breeds. The 1000 Bull Genomes Project provides a database of whole genome sequenced key ancestor bulls...

  2. Genomic prediction using QTL derived from whole genome sequence data

    DEFF Research Database (Denmark)

    Brøndum, Rasmus Froberg; Su, Guosheng; Janss, Luc

    This study investigated the gain in accuracy of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k SNP data. Analyses were performed for Nordic Holstein and Danish Jersey animals, using eithe...

  3. PREDICTION OF CHROMATIN STATES USING DNA SEQUENCE PROPERTIES

    KAUST Repository

    Bahabri, Rihab R.

    2013-06-01

    Activities of DNA are to a great extent controlled epigenetically through the internal struc- ture of chromatin. This structure is dynamic and is influenced by different modifications of histone proteins. Various combinations of epigenetic modification of histones pinpoint to different functional regions of the DNA determining the so-called chromatin states. How- ever, the characterization of chromatin states by the DNA sequence properties remains largely unknown. In this study we aim to explore whether DNA sequence patterns in the human genome can characterize different chromatin states. Using DNA sequence motifs we built binary classifiers for each chromatic state to eval- uate whether a given genomic sequence is a good candidate for belonging to a particular chromatin state. Of four classification algorithms (C4.5, Naive Bayes, Random Forest, and SVM) used for this purpose, the decision tree based classifiers (C4.5 and Random Forest) yielded best results among those we evaluated. Our results suggest that in general these models lack sufficient predictive power, although for four chromatin states (insulators, het- erochromatin, and two types of copy number variation) we found that presence of certain motifs in DNA sequences does imply an increased probability that such a sequence is one of these chromatin states.

  4. Multilocus Sequence Typing of Total-Genome-Sequenced Bacteria

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby; Cosentino, Salvatore; Rasmussen, Simon

    2012-01-01

    Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS...... the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56...... MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types...

  5. The characterization of twenty sequenced human genomes.

    Directory of Open Access Journals (Sweden)

    Kimberly Pelak

    2010-09-01

    Full Text Available We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

  6. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants.

    Science.gov (United States)

    Sucher, Nikolaus J; Hennell, James R; Carles, Maria C

    2012-01-01

    DNA fingerprinting of plants has become an invaluable tool in forensic, scientific, and industrial laboratories all over the world. PCR has become part of virtually every variation of the plethora of approaches used for DNA fingerprinting today. DNA sequencing is increasingly used either in combination with or as a replacement for traditional DNA fingerprinting techniques. A prime example is the use of short, standardized regions of the genome as taxon barcodes for biological identification of plants. Rapid advances in "next generation sequencing" (NGS) technology are driving down the cost of sequencing and bringing large-scale sequencing projects into the reach of individual investigators. We present an overview of recent publications that demonstrate the use of "NGS" technology for DNA fingerprinting and DNA barcoding applications.

  7. Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner

    DEFF Research Database (Denmark)

    Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan;

    2009-01-01

    MOTIVATION: The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary...

  8. Cloning and molecular genetics analyses of Deschampsia antarctica Desv. chloroplast and mitochondrial DNA sequence

    Directory of Open Access Journals (Sweden)

    O.P. Savchuk

    2012-03-01

    Full Text Available Chloroplast and mitochondrial DNA sequences of Deschampsia antarctica were studied. We had made comparison analysis with completely sequenced genomes of other temperateness plants to find homology.

  9. DNA secondary structures and epigenetic determinants of cancer genome evolution

    OpenAIRE

    2010-01-01

    An unstable genome is a hallmark of many cancers. It is unclear, however, whether some mutagenic features driving somatic alterations in cancer are encoded in the genome sequence and whether they can operate in a tissue-specific manner. We performed a genome-wide analysis of 663,446 DNA breakpoints associated with somatic copy-number alterations (SCNAs) from 2,792 cancer samples classified into 26 cancer types. Many SCNA breakpoints are spatially clustered in cancer genomes. We observed a sig...

  10. The mitochondrial genome sequence of the Tasmanian tiger (Thylacinus cynocephalus).

    Science.gov (United States)

    Miller, Webb; Drautz, Daniela I; Janecka, Jan E; Lesk, Arthur M; Ratan, Aakrosh; Tomsho, Lynn P; Packard, Mike; Zhang, Yeting; McClellan, Lindsay R; Qi, Ji; Zhao, Fangqing; Gilbert, M Thomas P; Dalén, Love; Arsuaga, Juan Luis; Ericson, Per G P; Huson, Daniel H; Helgen, Kristofer M; Murphy, William J; Götherström, Anders; Schuster, Stephan C

    2009-02-01

    We report the first two complete mitochondrial genome sequences of the thylacine (Thylacinus cynocephalus), or so-called Tasmanian tiger, extinct since 1936. The thylacine's phylogenetic position within australidelphian marsupials has long been debated, and here we provide strong support for the thylacine's basal position in Dasyuromorphia, aided by mitochondrial genome sequence that we generated from the extant numbat (Myrmecobius fasciatus). Surprisingly, both of our thylacine sequences differ by 11%-15% from putative thylacine mitochondrial genes in GenBank, with one of our samples originating from a direct offspring of the previously sequenced individual. Our data sample each mitochondrial nucleotide an average of 50 times, thereby providing the first high-fidelity reference sequence for thylacine population genetics. Our two sequences differ in only five nucleotides out of 15,452, hinting at a very low genetic diversity shortly before extinction. Despite the samples' heavy contamination with bacterial and human DNA and their temperate storage history, we estimate that as much as one-third of the total DNA in each sample is from the thylacine. The microbial content of the two thylacine samples was subjected to metagenomic analysis, and showed striking differences between a wild-captured individual and a born-in-captivity one. This study therefore adds to the growing evidence that extensive sequencing of museum collections is both feasible and desirable, and can yield complete genomes.

  11. Selective microbial genomic DNA isolation using restriction endonucleases.

    Science.gov (United States)

    Barnes, Helen E; Liu, Guohong; Weston, Christopher Q; King, Paula; Pham, Long K; Waltz, Shannon; Helzer, Kimberly T; Day, Laura; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

  12. Combining two technologies for full genome sequencing of human.

    Science.gov (United States)

    Skryabin, K G; Prokhortchouk, E B; Mazur, A M; Boulygina, E S; Tsygankova, S V; Nedoluzhko, A V; Rastorguev, S M; Matveev, V B; Chekanov, N N; D A, Goranskaya; Teslyuk, A B; Gruzdeva, N M; Velikhov, V E; Zaridze, D G; Kovalchuk, M V

    2009-10-01

    At present, the new technologies of DNA sequencing are rapidly developing allowing quick and efficient characterisation of organisms at the level of the genome structure. In this study, the whole genome sequencing of a human (Russian man) was performed using two technologies currently present on the market - Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) (Applied Biosystems) and sequencing technologies of molecular clusters using fluorescently labeled precursors (Illumina). The total number of generated data resulted in 108.3 billion base pairs (60.2 billion from Illumina technology and 48.1 billion from SOLiD technology). Statistics performed on reads generated by GAII and SOLiD showed that they covered 75% and 96% of the genome respectively. Short polymorphic regions were detected with comparable accuracy however, the absolute amount of them revealed by SOLiD was several times less than by GAII. Optimal algorithm for using the latest methods of sequencing was established for the analysis of individual human genomes. The study is the first Russian effort towards whole human genome sequencing.

  13. 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.

  14. Specialized microbial databases for inductive exploration of microbial genome sequences

    Directory of Open Access Journals (Sweden)

    Cabau Cédric

    2005-02-01

    Full Text Available Abstract Background The enormous amount of genome sequence data asks for user-oriented databases to manage sequences and annotations. Queries must include search tools permitting function identification through exploration of related objects. Methods The GenoList package for collecting and mining microbial genome databases has been rewritten using MySQL as the database management system. Functions that were not available in MySQL, such as nested subquery, have been implemented. Results Inductive reasoning in the study of genomes starts from "islands of knowledge", centered around genes with some known background. With this concept of "neighborhood" in mind, a modified version of the GenoList structure has been used for organizing sequence data from prokaryotic genomes of particular interest in China. GenoChore http://bioinfo.hku.hk/genochore.html, a set of 17 specialized end-user-oriented microbial databases (including one instance of Microsporidia, Encephalitozoon cuniculi, a member of Eukarya has been made publicly available. These databases allow the user to browse genome sequence and annotation data using standard queries. In addition they provide a weekly update of searches against the world-wide protein sequences data libraries, allowing one to monitor annotation updates on genes of interest. Finally, they allow users to search for patterns in DNA or protein sequences, taking into account a clustering of genes into formal operons, as well as providing extra facilities to query sequences using predefined sequence patterns. Conclusion This growing set of specialized microbial databases organize data created by the first Chinese bacterial genome programs (ThermaList, Thermoanaerobacter tencongensis, LeptoList, with two different genomes of Leptospira interrogans and SepiList, Staphylococcus epidermidis associated to related organisms for comparison.

  15. First fungal genome sequence from Africa: A preliminary analysis

    Directory of Open Access Journals (Sweden)

    Rene Sutherland

    2012-01-01

    Full Text Available Some of the most significant breakthroughs in the biological sciences this century will emerge from the development of next generation sequencing technologies. The ease of availability of DNA sequence made possible through these new technologies has given researchers opportunities to study organisms in a manner that was not possible with Sanger sequencing. Scientists will, therefore, need to embrace genomics, as well as develop and nurture the human capacity to sequence genomes and utilise the ’tsunami‘ of data that emerge from genome sequencing. In response to these challenges, we sequenced the genome of Fusarium circinatum, a fungal pathogen of pine that causes pitch canker, a disease of great concern to the South African forestry industry. The sequencing work was conducted in South Africa, making F. circinatum the first eukaryotic organism for which the complete genome has been sequenced locally. Here we report on the process that was followed to sequence, assemble and perform a preliminary characterisation of the genome. Furthermore, details of the computer annotation and manual curation of this genome are presented. The F. circinatum genome was found to be nearly 44 million bases in size, which is similar to that of four other Fusarium genomes that have been sequenced elsewhere. The genome contains just over 15 000 open reading frames, which is less than that of the related species, Fusarium oxysporum, but more than that for Fusarium verticillioides. Amongst the various putative gene clusters identified in F. circinatum, those encoding the secondary metabolites fumosin and fusarin appeared to harbour evidence of gene translocation. It is anticipated that similar comparisons of other loci will provide insights into the genetic basis for pathogenicity of the pitch canker pathogen. Perhaps more importantly, this project has engaged a relatively large group of scientists

  16. Similarity Estimation Between DNA Sequences Based on Local Pattern Histograms of Binary Images

    Institute of Scientific and Technical Information of China (English)

    Yusei Kobori; Satoshi Mizuta

    2016-01-01

    Graphical representation of DNA sequences is one of the most popular techniques for alignment-free sequence comparison. Here, we propose a new method for the feature extraction of DNA sequences represented by binary images, by estimating the similarity between DNA sequences using the frequency histograms of local bitmap patterns of images. Our method shows linear time complexity for the length of DNA sequences, which is practical even when long sequences, such as whole genome sequences, are compared. We tested five distance measures for the estimation of sequence similarities, and found that the histogram intersection and Manhattan distance are the most appropriate ones for phylogenetic analyses.

  17. Draft genome sequence of the rubber tree Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    Rahman Ahmad Yamin Abdul

    2013-02-01

    Full Text Available Abstract Background Hevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR. NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876. Results Here, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified. Conclusions The knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber.

  18. Whole genome sequencing in clinical and public health microbiology.

    Science.gov (United States)

    Kwong, J C; McCallum, N; Sintchenko, V; Howden, B P

    2015-04-01

    Genomics and whole genome sequencing (WGS) have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology.The growth and availability of bench-top WGS analysers has facilitated the feasibility of genomics in clinical and public health microbiology.Given current resource and infrastructure limitations, WGS is most applicable to use in public health laboratories, reference laboratories, and hospital infection control-affiliated laboratories.As WGS represents the pinnacle for strain characterisation and epidemiological analyses, it is likely to replace traditional typing methods, resistance gene detection and other sequence-based investigations (e.g., 16S rDNA PCR) in the near future.Although genomic technologies are rapidly evolving, widespread implementation in clinical and public health microbiology laboratories is limited by the need for effective semi-automated pipelines, standardised quality control and data interpretation, bioinformatics expertise, and infrastructure.

  19. Nanopore-based Fourth-generation DNA Sequencing Technology

    Institute of Scientific and Technical Information of China (English)

    Yanxiao Feng; Yuechuan Zhang; Cuifeng Ying; Deqiang Wang; Chunlei Du

    2015-01-01

    Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than$100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.

  20. Microsatellite discovery by deep sequencing of enriched genomic libraries.

    Science.gov (United States)

    Santana, Quentin; Coetzee, Martin; Steenkamp, Emma; Mlonyeni, Osmond; Hammond, Gifty; Wingfield, Michael; Wingfield, Brenda

    2009-03-01

    Robust molecular markers such as microsatellites are important tools used to understand the dynamics of natural populations, but their identification and development are typically time consuming and labor intensive. The recent emergence of so-called next-generation sequencing raised the question as to whether this new technology might be applied to microsatellite development. Following this view, we considered whether deep sequencing using the 454 Life Sciences/Roche GS-FLX genome sequencing system could lead to a rapid protocol to develop microsatellite primers as markers for genetic studies. For this purpose, genomic DNA was sourced from three unrelated organisms: a fungus (the pine pathogen Fusarium circinatum), an insect (the pine-damaging wasp Sirex noctilio), and the wasp's associated nematode parasite (Deladenus siricidicola). Two methods, FIASCO (fast isolation by AFLP of sequences containing repeats) and ISSR-PCR (inter-simple sequence repeat PCR), were used to generate microsatellite-enriched DNA for the 454 libraries. From the resulting 1.2-1.7 megabases of DNA sequence data, we were able to identify 873 microsatellites that have sufficient flanking sequence available for primer design and potential amplification. This approach to microsatellite discovery was substantially more rapid, effective, and economical than other methods, and this study has shown that pyrosequencing provides an outstanding new technology that can be applied to this purpose.

  1. The roles of adenoviral vectors and donor DNA structures on genome editing

    NARCIS (Netherlands)

    Holkers, Maarten

    2016-01-01

    Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign sequen

  2. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

  3. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2011-01-01

    43101491 SNP. Following adjustment for multiple-testing, significant association (q ≤ 0.05 remained between the rs41694646 SNP and four traits (animal stature, body depth, direct calving difficulty and milk yield only. Notably, the single SNP in the bovine NESP55 gene (rs41694656 was associated (P ≤ 0.01 with somatic cell count--an often-cited indicator of resistance to mastitis and overall health status of the mammary system--and previous studies have demonstrated that the chromosomal region to where the GNAS domain maps underlies an important quantitative trait locus for this trait. This association, however, was not significant after adjustment for multiple testing. The three remaining SNPs assayed were not associated with any of the performance traits analysed in this study. Analysis of all pairwise linkage disequilibrium (r2 values suggests that most allele substitution effects for the assayed SNPs observed are independent. Finally, the polymorphic coding SNP in the putative bovine NESP55 gene was used to test the imprinting status of this gene across a range of foetal bovine tissues. Conclusions Previous studies in other mammalian species have shown that DNA sequence variation within the imprinted GNAS gene cluster contributes to several physiological and metabolic disorders, including obesity in humans and mice. Similarly, the results presented here indicate an important role for the imprinted GNAS cluster in underlying complex performance traits in cattle such as animal growth, calving, fertility and health. These findings suggest that GNAS domain-associated polymorphisms may serve as important genetic markers for future livestock breeding programs and support previous studies that candidate imprinted loci may act as molecular targets for the genetic improvement of agricultural populations. In addition, we present new evidence that the bovine NESP55 gene is epigenetically regulated as a maternally expressed imprinted gene in placental and intestinal tissues

  4. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits

    Science.gov (United States)

    2011-01-01

    adjustment for multiple-testing, significant association (q ≤ 0.05) remained between the rs41694646 SNP and four traits (animal stature, body depth, direct calving difficulty and milk yield) only. Notably, the single SNP in the bovine NESP55 gene (rs41694656) was associated (P ≤ 0.01) with somatic cell count--an often-cited indicator of resistance to mastitis and overall health status of the mammary system--and previous studies have demonstrated that the chromosomal region to where the GNAS domain maps underlies an important quantitative trait locus for this trait. This association, however, was not significant after adjustment for multiple testing. The three remaining SNPs assayed were not associated with any of the performance traits analysed in this study. Analysis of all pairwise linkage disequilibrium (r2) values suggests that most allele substitution effects for the assayed SNPs observed are independent. Finally, the polymorphic coding SNP in the putative bovine NESP55 gene was used to test the imprinting status of this gene across a range of foetal bovine tissues. Conclusions Previous studies in other mammalian species have shown that DNA sequence variation within the imprinted GNAS gene cluster contributes to several physiological and metabolic disorders, including obesity in humans and mice. Similarly, the results presented here indicate an important role for the imprinted GNAS cluster in underlying complex performance traits in cattle such as animal growth, calving, fertility and health. These findings suggest that GNAS domain-associated polymorphisms may serve as important genetic markers for future livestock breeding programs and support previous studies that candidate imprinted loci may act as molecular targets for the genetic improvement of agricultural populations. In addition, we present new evidence that the bovine NESP55 gene is epigenetically regulated as a maternally expressed imprinted gene in placental and intestinal tissues from 8-10 week old

  5. Complete genome sequence of arracacha mottle virus.

    Science.gov (United States)

    Orílio, Anelise F; Lucinda, Natalia; Dusi, André N; Nagata, Tatsuya; Inoue-Nagata, Alice K

    2013-01-01

    Arracacha mottle virus (AMoV) is the only potyvirus reported to infect arracacha (Arracacia xanthorrhiza) in Brazil. Here, the complete genome sequence of an isolate of AMoV was determined to be 9,630 nucleotides in length, excluding the 3' poly-A tail, and encoding a polyprotein of 3,135 amino acids and a putative P3N-PIPO protein. Its genomic organization is typical of a member of the genus Potyvirus, containing all conserved motifs. Its full genome sequence shared 56.2 % nucleotide identity with sunflower chlorotic mottle virus and verbena virus Y, the most closely related viruses.

  6. Design optimization methods for genomic DNA tiling arrays.

    Science.gov (United States)

    Bertone, Paul; Trifonov, Valery; Rozowsky, Joel S; Schubert, Falk; Emanuelsson, Olof; Karro, John; Kao, Ming-Yang; Snyder, Michael; Gerstein, Mark

    2006-02-01

    A recent development in microarray research entails the unbiased coverage, or tiling, of genomic DNA for the large-scale identification of transcribed sequences and regulatory elements. A central issue in designing tiling arrays is that of arriving at a single-copy tile path, as significant sequence cross-hybridization can result from the presence of non-unique probes on the array. Due to the fragmentation of genomic DNA caused by the widespread distribution of repetitive elements, the problem of obtaining adequate sequence coverage increases with the sizes of subsequence tiles that are to be included in the design. This becomes increasingly problematic when considering complex eukaryotic genomes that contain many thousands of interspersed repeats. The general problem of sequence tiling can be framed as finding an optimal partitioning of non-repetitive subsequences over a prescribed range of tile sizes, on a DNA sequence comprising repetitive and non-repetitive regions. Exact solutions to the tiling problem become computationally infeasible when applied to large genomes, but successive optimizations are developed that allow their practical implementation. These include an efficient method for determining the degree of similarity of many oligonucleotide sequences over large genomes, and two algorithms for finding an optimal tile path composed of longer sequence tiles. The first algorithm, a dynamic programming approach, finds an optimal tiling in linear time and space; the second applies a heuristic search to reduce the space complexity to a constant requirement. A Web resource has also been developed, accessible at http://tiling.gersteinlab.org, to generate optimal tile paths from user-provided DNA sequences.

  7. Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements

    Directory of Open Access Journals (Sweden)

    Pareja Eduardo

    2006-03-01

    Full Text Available Abstract Background Mobile elements are involved in genomic rearrangements and virulence acquisition, and hence, are important elements in bacterial genome evolution. The insertion of some specific Insertion Sequences had been associated with repetitive extragenic palindromic (REP elements. Considering that there are a sufficient number of available genomes with described REPs, and exploiting the advantage of the traceability of transposition events in genomes, we decided to exhaustively analyze the relationship between REP sequences and mobile elements. Results This global multigenome study highlights the importance of repetitive extragenic palindromic elements as target sequences for transposases. The study is based on the analysis of the DNA regions surrounding the 981 instances of Insertion Sequence elements with respect to the positioning of REP sequences in the 19 available annotated microbial genomes corresponding to species of bacteria with reported REP sequences. This analysis has allowed the detection of the specific insertion into REP sequences for ISPsy8 in Pseudomonas syringae DC3000, ISPa11 in P. aeruginosa PA01, ISPpu9 and ISPpu10 in P. putida KT2440, and ISRm22 and ISRm19 in Sinorhizobium meliloti 1021 genome. Preference for insertion in extragenic spaces with REP sequences has also been detected for ISPsy7 in P. syringae DC3000, ISRm5 in S. meliloti and ISNm1106 in Neisseria meningitidis MC58 and Z2491 genomes. Probably, the association with REP elements that we have detected analyzing genomes is only the tip of the iceberg, and this association could be even more frequent in natural isolates. Conclusion Our findings characterize REP elements as hot spots for transposition and reinforce the relationship between REP sequences and genomic plasticity mediated by mobile elements. In addition, this study defines a subset of REP-recognizer transposases with high target selectivity that can be useful in the development of new tools for

  8. A complete mitochondrial genome sequence from a mesolithic wild aurochs (Bos primigenius).

    LENUS (Irish Health Repository)

    Edwards, Ceiridwen J

    2010-01-01

    BACKGROUND: The derivation of domestic cattle from the extinct wild aurochs (Bos primigenius) has been well-documented by archaeological and genetic studies. Genetic studies point towards the Neolithic Near East as the centre of origin for Bos taurus, with some lines of evidence suggesting possible, albeit rare, genetic contributions from locally domesticated wild aurochsen across Eurasia. Inferences from these investigations have been based largely on the analysis of partial mitochondrial DNA sequences generated from modern animals, with limited sequence data from ancient aurochsen samples. Recent developments in DNA sequencing technologies, however, are affording new opportunities for the examination of genetic material retrieved from extinct species, providing new insight into their evolutionary history. Here we present DNA sequence analysis of the first complete mitochondrial genome (16,338 base pairs) from an archaeologically-verified and exceptionally-well preserved aurochs bone sample. METHODOLOGY: DNA extracts were generated from an aurochs humerus bone sample recovered from a cave site located in Derbyshire, England and radiocarbon-dated to 6,738+\\/-68 calibrated years before present. These extracts were prepared for both Sanger and next generation DNA sequencing technologies (Illumina Genome Analyzer). In total, 289.9 megabases (22.48%) of the post-filtered DNA sequences generated using the Illumina Genome Analyzer from this sample mapped with confidence to the bovine genome. A consensus B. primigenius mitochondrial genome sequence was constructed and was analysed alongside all available complete bovine mitochondrial genome sequences. CONCLUSIONS: For all nucleotide positions where both Sanger and Illumina Genome Analyzer sequencing methods gave high-confidence calls, no discrepancies were observed. Sequence analysis reveals evidence of heteroplasmy in this sample and places this mitochondrial genome seque