Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach.

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Shota Nakamura

Full Text Available With the severe acute respiratory syndrome epidemic of 2003 and renewed attention on avian influenza viral pandemics, new surveillance systems are needed for the earlier detection of emerging infectious diseases. We applied a "next-generation" parallel sequencing platform for viral detection in nasopharyngeal and fecal samples collected during seasonal influenza virus (Flu infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.1-0.25 ml of nasopharyngeal aspirates (N = 3 and fecal specimens (N = 5, and more than 10 microg of cDNA was synthesized. Unbiased high-throughput sequencing of these 8 samples yielded 15,298-32,335 (average 24,738 reads in a single 7.5 h run. In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90% of reads were host genome-derived, 20-460 Flu-reads were detected, which was sufficient for subtype identification. In fecal samples, bacteria and host cells were removed by centrifugation, resulting in gain of 484-15,260 reads of norovirus sequence (78-98% of the whole genome was covered, except for one specimen that was under-detectable by RT-PCR. These results suggest that our unbiased high-throughput sequencing approach is useful for directly detecting pathogenic viruses without advance genetic information. Although its cost and technological availability make it unlikely that this system will very soon be the diagnostic standard worldwide, this system could be useful for the earlier discovery of novel emerging viruses and bioterrorism, which are difficult to detect with conventional procedures.

High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

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Christiansen, Anders; Kringelum, Jens Vindahl; Hansen, Christian Skjødt

2015-01-01

of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage...

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

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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...... that the data produced is optimal. Although much of the procedure can be followed directly from the manufacturer's protocols, the key differences lie in the library preparation steps. This chapter presents an optimized protocol for the sequencing of fossil remains and museum specimens, commonly referred...

High-throughput Sequencing Based Immune Repertoire Study during Infectious Disease

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Dongni Hou

2016-08-01

Full Text Available The selectivity of the adaptive immune response is based on the enormous diversity of T and B cell antigen-specific receptors. The immune repertoire, the collection of T and B cells with functional diversity in the circulatory system at any given time, is dynamic and reflects the essence of immune selectivity. In this article, we review the recent advances in immune repertoire study of infectious diseases that achieved by traditional techniques and high-throughput sequencing techniques. High-throughput sequencing techniques enable the determination of complementary regions of lymphocyte receptors with unprecedented efficiency and scale. This progress in methodology enhances the understanding of immunologic changes during pathogen challenge, and also provides a basis for further development of novel diagnostic markers, immunotherapies and vaccines.

Application of high-throughput DNA sequencing in phytopathology.

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Studholme, David J; Glover, Rachel H; Boonham, Neil

2011-01-01

The new sequencing technologies are already making a big impact in academic research on medically important microbes and may soon revolutionize diagnostics, epidemiology, and infection control. Plant pathology also stands to gain from exploiting these opportunities. This manuscript reviews some applications of these high-throughput sequencing methods that are relevant to phytopathology, with emphasis on the associated computational and bioinformatics challenges and their solutions. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses. Copyright © 2011 by Annual Reviews. All rights reserved.

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

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Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M; Bloom, David C; McIntyre, Lauren M

2017-08-16

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

Sources of PCR-induced distortions in high-throughput sequencing data sets

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Kebschull, Justus M.; Zador, Anthony M.

2015-01-01

PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

On the optimal trimming of high-throughput mRNA sequence data

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Matthew D MacManes

2014-01-01

Full Text Available The widespread and rapid adoption of high-throughput sequencing technologies has afforded researchers the opportunity to gain a deep understanding of genome level processes that underlie evolutionary change, and perhaps more importantly, the links between genotype and phenotype. In particular, researchers interested in functional biology and adaptation have used these technologies to sequence mRNA transcriptomes of specific tissues, which in turn are often compared to other tissues, or other individuals with different phenotypes. While these techniques are extremely powerful, careful attention to data quality is required. In particular, because high-throughput sequencing is more error-prone than traditional Sanger sequencing, quality trimming of sequence reads should be an important step in all data processing pipelines. While several software packages for quality trimming exist, no general guidelines for the specifics of trimming have been developed. Here, using empirically derived sequence data, I provide general recommendations regarding the optimal strength of trimming, specifically in mRNA-Seq studies. Although very aggressive quality trimming is common, this study suggests that a more gentle trimming, specifically of those nucleotides whose Phred score < 2 or < 5, is optimal for most studies across a wide variety of metrics.

Analysis of high-throughput sequencing and annotation strategies for phage genomes.

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Matthew R Henn

Full Text Available BACKGROUND: Bacterial viruses (phages play a critical role in shaping microbial populations as they influence both host mortality and horizontal gene transfer. As such, they have a significant impact on local and global ecosystem function and human health. Despite their importance, little is known about the genomic diversity harbored in phages, as methods to capture complete phage genomes have been hampered by the lack of knowledge about the target genomes, and difficulties in generating sufficient quantities of genomic DNA for sequencing. Of the approximately 550 phage genomes currently available in the public domain, fewer than 5% are marine phage. METHODOLOGY/PRINCIPAL FINDINGS: To advance the study of phage biology through comparative genomic approaches we used marine cyanophage as a model system. We compared DNA preparation methodologies (DNA extraction directly from either phage lysates or CsCl purified phage particles, and sequencing strategies that utilize either Sanger sequencing of a linker amplification shotgun library (LASL or of a whole genome shotgun library (WGSL, or 454 pyrosequencing methods. We demonstrate that genomic DNA sample preparation directly from a phage lysate, combined with 454 pyrosequencing, is best suited for phage genome sequencing at scale, as this method is capable of capturing complete continuous genomes with high accuracy. In addition, we describe an automated annotation informatics pipeline that delivers high-quality annotation and yields few false positives and negatives in ORF calling. CONCLUSIONS/SIGNIFICANCE: These DNA preparation, sequencing and annotation strategies enable a high-throughput approach to the burgeoning field of phage genomics.

High Throughput Sequencing for Detection of Foodborne Pathogens

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Camilla Sekse

2017-10-01

Full Text Available High-throughput sequencing (HTS is becoming the state-of-the-art technology for typing of microbial isolates, especially in clinical samples. Yet, its application is still in its infancy for monitoring and outbreak investigations of foods. Here we review the published literature, covering not only bacterial but also viral and Eukaryote food pathogens, to assess the status and potential of HTS implementation to inform stakeholders, improve food safety and reduce outbreak impacts. The developments in sequencing technology and bioinformatics have outpaced the capacity to analyze and interpret the sequence data. The influence of sample processing, nucleic acid extraction and purification, harmonized protocols for generation and interpretation of data, and properly annotated and curated reference databases including non-pathogenic “natural” strains are other major obstacles to the realization of the full potential of HTS in analytical food surveillance, epidemiological and outbreak investigations, and in complementing preventive approaches for the control and management of foodborne pathogens. Despite significant obstacles, the achieved progress in capacity and broadening of the application range over the last decade is impressive and unprecedented, as illustrated with the chosen examples from the literature. Large consortia, often with broad international participation, are making coordinated efforts to cope with many of the mentioned obstacles. Further rapid progress can therefore be prospected for the next decade.

Assessing the Diversity of Rodent-Borne Viruses: Exploring of High-Throughput Sequencing and Classical Amplification/Sequencing Approaches.

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Drewes, Stephan; Straková, Petra; Drexler, Jan F; Jacob, Jens; Ulrich, Rainer G

2017-01-01

Rodents are distributed throughout the world and interact with humans in many ways. They provide vital ecosystem services, some species are useful models in biomedical research and some are held as pet animals. However, many rodent species can have adverse effects such as damage to crops and stored produce, and they are of health concern because of the transmission of pathogens to humans and livestock. The first rodent viruses were discovered by isolation approaches and resulted in break-through knowledge in immunology, molecular and cell biology, and cancer research. In addition to rodent-specific viruses, rodent-borne viruses are causing a large number of zoonotic diseases. Most prominent examples are reemerging outbreaks of human hemorrhagic fever disease cases caused by arena- and hantaviruses. In addition, rodents are reservoirs for vector-borne pathogens, such as tick-borne encephalitis virus and Borrelia spp., and may carry human pathogenic agents, but likely are not involved in their transmission to human. In our days, next-generation sequencing or high-throughput sequencing (HTS) is revolutionizing the speed of the discovery of novel viruses, but other molecular approaches, such as generic RT-PCR/PCR and rolling circle amplification techniques, contribute significantly to the rapidly ongoing process. However, the current knowledge still represents only the tip of the iceberg, when comparing the known human viruses to those known for rodents, the mammalian taxon with the largest species number. The diagnostic potential of HTS-based metagenomic approaches is illustrated by their use in the discovery and complete genome determination of novel borna- and adenoviruses as causative disease agents in squirrels. In conclusion, HTS, in combination with conventional RT-PCR/PCR-based approaches, resulted in a drastically increased knowledge of the diversity of rodent viruses. Future improvements of the used workflows, including bioinformatics analysis, will further

Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

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Randi Holm Jensen

Full Text Available Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.

SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes.

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Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

2012-07-15

In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license.

eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing.

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Yuan, Tiezheng; Huang, Xiaoyi; Dittmar, Rachel L; Du, Meijun; Kohli, Manish; Boardman, Lisa; Thibodeau, Stephen N; Wang, Liang

2014-03-05

RNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers. We developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification" includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module "mRNA identification" includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module "Target screening" provides expression profiling analyses and graphic visualization. The module "Self-testing" offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program's functionality. eRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory.

Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing

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Zhang, Xiao-Yong; Wang, Guang-Hua; Xu, Xin-Ya; Nong, Xu-Hua; Wang, Jie; Amin, Muhammad; Qi, Shu-Hua

2016-10-01

The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deep-sea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing.

Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

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Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

2014-01-01

High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

Galaxy Workflows for Web-based Bioinformatics Analysis of Aptamer High-throughput Sequencing Data

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William H Thiel

2016-01-01

Full Text Available Development of RNA and DNA aptamers for diagnostic and therapeutic applications is a rapidly growing field. Aptamers are identified through iterative rounds of selection in a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment. High-throughput sequencing (HTS revolutionized the modern SELEX process by identifying millions of aptamer sequences across multiple rounds of aptamer selection. However, these vast aptamer HTS datasets necessitated bioinformatics techniques. Herein, we describe a semiautomated approach to analyze aptamer HTS datasets using the Galaxy Project, a web-based open source collection of bioinformatics tools that were originally developed to analyze genome, exome, and transcriptome HTS data. Using a series of Workflows created in the Galaxy webserver, we demonstrate efficient processing of aptamer HTS data and compilation of a database of unique aptamer sequences. Additional Workflows were created to characterize the abundance and persistence of aptamer sequences within a selection and to filter sequences based on these parameters. A key advantage of this approach is that the online nature of the Galaxy webserver and its graphical interface allow for the analysis of HTS data without the need to compile code or install multiple programs.

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

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Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data.

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Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

2010-07-01

High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users.

Quack: A quality assurance tool for high throughput sequence data.

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Thrash, Adam; Arick, Mark; Peterson, Daniel G

2018-05-01

The quality of data generated by high-throughput DNA sequencing tools must be rapidly assessed in order to determine how useful the data may be in making biological discoveries; higher quality data leads to more confident results and conclusions. Due to the ever-increasing size of data sets and the importance of rapid quality assessment, tools that analyze sequencing data should quickly produce easily interpretable graphics. Quack addresses these issues by generating information-dense visualizations from FASTQ files at a speed far surpassing other publicly available quality assurance tools in a manner independent of sequencing technology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing

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Li Kelvin

2012-11-01

Full Text Available Abstract Background In a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally. Results We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute’s (JCVI high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates. Conclusions Augmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus

Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

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Trujillano, Daniel; Perez, Belén; González, Justo; Tornador, Cristian; Navarrete, Rosa; Escaramis, Georgia; Ossowski, Stephan; Armengol, Lluís; Cornejo, Verónica; Desviat, Lourdes R; Ugarte, Magdalena; Estivill, Xavier

2014-01-01

Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth. PMID:23942198

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

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Mullen Michael P

2012-01-01

Full Text Available Abstract Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952 of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612 were intronic and 9% (n = 464 were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS. Significant (P ® MassARRAY. No significant differences (P > 0.1 were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total. Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving

Combining Amplification Typing of L1 Active Subfamilies (ATLAS) with High-Throughput Sequencing.

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Rahbari, Raheleh; Badge, Richard M

2016-01-01

With the advent of new generations of high-throughput sequencing technologies, the catalog of human genome variants created by retrotransposon activity is expanding rapidly. However, despite these advances in describing L1 diversity and the fact that L1 must retrotranspose in the germline or prior to germline partitioning to be evolutionarily successful, direct assessment of de novo L1 retrotransposition in the germline or early embryogenesis has not been achieved for endogenous L1 elements. A direct study of de novo L1 retrotransposition into susceptible loci within sperm DNA (Freeman et al., Hum Mutat 32(8):978-988, 2011) suggested that the rate of L1 retrotransposition in the germline is much lower than previously estimated (ATLAS L1 display technique (Badge et al., Am J Hum Genet 72(4):823-838, 2003) to investigate de novo L1 retrotransposition in human genomes. In this chapter, we describe how we combined a high-coverage ATLAS variant with high-throughput sequencing, achieving 11-25× sequence depth per single amplicon, to study L1 retrotransposition in whole genome amplified (WGA) DNAs.

Improvements and impacts of GRCh38 human reference on high throughput sequencing data analysis.

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Guo, Yan; Dai, Yulin; Yu, Hui; Zhao, Shilin; Samuels, David C; Shyr, Yu

2017-03-01

Analyses of high throughput sequencing data starts with alignment against a reference genome, which is the foundation for all re-sequencing data analyses. Each new release of the human reference genome has been augmented with improved accuracy and completeness. It is presumed that the latest release of human reference genome, GRCh38 will contribute more to high throughput sequencing data analysis by providing more accuracy. But the amount of improvement has not yet been quantified. We conducted a study to compare the genomic analysis results between the GRCh38 reference and its predecessor GRCh37. Through analyses of alignment, single nucleotide polymorphisms, small insertion/deletions, copy number and structural variants, we show that GRCh38 offers overall more accurate analysis of human sequencing data. More importantly, GRCh38 produced fewer false positive structural variants. In conclusion, GRCh38 is an improvement over GRCh37 not only from the genome assembly aspect, but also yields more reliable genomic analysis results. Copyright © 2017. Published by Elsevier Inc.

High-Throughput DNA sequencing of ancient wood.

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Wagner, Stefanie; Lagane, Frédéric; Seguin-Orlando, Andaine; Schubert, Mikkel; Leroy, Thibault; Guichoux, Erwan; Chancerel, Emilie; Bech-Hebelstrup, Inger; Bernard, Vincent; Billard, Cyrille; Billaud, Yves; Bolliger, Matthias; Croutsch, Christophe; Čufar, Katarina; Eynaud, Frédérique; Heussner, Karl Uwe; Köninger, Joachim; Langenegger, Fabien; Leroy, Frédéric; Lima, Christine; Martinelli, Nicoletta; Momber, Garry; Billamboz, André; Nelle, Oliver; Palomo, Antoni; Piqué, Raquel; Ramstein, Marianne; Schweichel, Roswitha; Stäuble, Harald; Tegel, Willy; Terradas, Xavier; Verdin, Florence; Plomion, Christophe; Kremer, Antoine; Orlando, Ludovic

2018-03-01

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high-throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long-term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human-induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro-evolutionary response of trees to climate change and human forest management. © 2018 John Wiley & Sons Ltd.

Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

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Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

2018-03-09

High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

Science.gov (United States)

Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

2017-04-15

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

Science.gov (United States)

Hu, Jiazhi; Meyers, Robin M; Dong, Junchao; Panchakshari, Rohit A; Alt, Frederick W; Frock, Richard L

2016-05-01

Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

Discovery of viruses and virus-like pathogens in pistachio using high-throughput sequencing

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Pistachio (Pistacia vera L.) trees from the National Clonal Germplasm Repository (NCGR) and orchards in California were surveyed for viruses and virus-like agents by high-throughput sequencing (HTS). Analyses of 60 trees including clonal UCB-1 hybrid rootstock (P. atlantica × P. integerrima) identif...

Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

OpenAIRE

MORRILL, Benson H.; COX, Lindsay; WARD, Anika; HEYWOOD, Sierra; PRATHER, Randall S.; ISOM, S. Clay

2013-01-01

Abstract The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx plat...

High-throughput sequencing of forensic genetic samples using punches of FTA cards with buccal swabs

DEFF Research Database (Denmark)

Kampmann, Marie-Louise; Buchard, Anders; Børsting, Claus

2016-01-01

Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards...

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.

Determining the diet of larvae of western rock lobster (Panulirus cygnus using high-throughput DNA sequencing techniques.

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Richard O'Rorke

Full Text Available The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea, Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut. High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of lobster larvae.

SUGAR: graphical user interface-based data refiner for high-throughput DNA sequencing.

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Sato, Yukuto; Kojima, Kaname; Nariai, Naoki; Yamaguchi-Kabata, Yumi; Kawai, Yosuke; Takahashi, Mamoru; Mimori, Takahiro; Nagasaki, Masao

2014-08-08

Next-generation sequencers (NGSs) have become one of the main tools for current biology. To obtain useful insights from the NGS data, it is essential to control low-quality portions of the data affected by technical errors such as air bubbles in sequencing fluidics. We develop a software SUGAR (subtile-based GUI-assisted refiner) which can handle ultra-high-throughput data with user-friendly graphical user interface (GUI) and interactive analysis capability. The SUGAR generates high-resolution quality heatmaps of the flowcell, enabling users to find possible signals of technical errors during the sequencing. The sequencing data generated from the error-affected regions of a flowcell can be selectively removed by automated analysis or GUI-assisted operations implemented in the SUGAR. The automated data-cleaning function based on sequence read quality (Phred) scores was applied to a public whole human genome sequencing data and we proved the overall mapping quality was improved. The detailed data evaluation and cleaning enabled by SUGAR would reduce technical problems in sequence read mapping, improving subsequent variant analysis that require high-quality sequence data and mapping results. Therefore, the software will be especially useful to control the quality of variant calls to the low population cells, e.g., cancers, in a sample with technical errors of sequencing procedures.

Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing

DEFF Research Database (Denmark)

Vinner, Lasse; Mourier, Tobias; Friis-Nielsen, Jens

2015-01-01

-stringency in-solution hybridization method enables detection of discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral...... sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer...

The main challenges that remain in applying high-throughput sequencing to clinical diagnostics.

Science.gov (United States)

Loeffelholz, Michael; Fofanov, Yuriy

2015-01-01

Over the last 10 years, the quality, price and availability of high-throughput sequencing instruments have improved to the point that this technology may be close to becoming a routine tool in the diagnostic microbiology laboratory. Two groups of challenges, however, have to be resolved in order to move this powerful research technology into routine use in the clinical microbiology laboratory. The computational/bioinformatics challenges include data storage cost and privacy concerns, requiring analysis to be performed without access to cloud storage or expensive computational infrastructure. The logistical challenges include interpretation of complex results and acceptance and understanding of the advantages and limitations of this technology by the medical community. This article focuses on the approaches to address these challenges, such as file formats, algorithms, data collection, reporting and good laboratory practices.

ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data.

Science.gov (United States)

Luo, Guan-Zheng; Yang, Wei; Ma, Ying-Ke; Wang, Xiu-Jie

2014-02-01

Integrative Short Reads NAvigator (ISRNA) is an online toolkit for analyzing high-throughput small RNA sequencing data. Besides the high-speed genome mapping function, ISRNA provides statistics for genomic location, length distribution and nucleotide composition bias analysis of sequence reads. Number of reads mapped to known microRNAs and other classes of short non-coding RNAs, coverage of short reads on genes, expression abundance of sequence reads as well as some other analysis functions are also supported. The versatile search functions enable users to select sequence reads according to their sub-sequences, expression abundance, genomic location, relationship to genes, etc. A specialized genome browser is integrated to visualize the genomic distribution of short reads. ISRNA also supports management and comparison among multiple datasets. ISRNA is implemented in Java/C++/Perl/MySQL and can be freely accessed at http://omicslab.genetics.ac.cn/ISRNA/.

High-throughput sequencing approach uncovers the miRNome of peritoneal endometriotic lesions and adjacent healthy tissues.

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Merli Saare

Full Text Available Accumulating data have shown the involvement of microRNAs (miRNAs in endometriosis pathogenesis. In this study, we used a novel approach to determine the endometriotic lesion-specific miRNAs by high-throughput small RNA sequencing of paired samples of peritoneal endometriotic lesions and matched healthy surrounding tissues together with eutopic endometria of the same patients. We found five miRNAs specific to epithelial cells--miR-34c, miR-449a, miR-200a, miR-200b and miR-141 showing significantly higher expression in peritoneal endometriotic lesions compared to healthy peritoneal tissues. We also determined the expression levels of miR-200 family target genes E-cadherin, ZEB1 and ZEB2 and found that the expression level of E-cadherin was significantly higher in endometriotic lesions compared to healthy tissues. Further evaluation verified that studied miRNAs could be used as diagnostic markers for confirming the presence of endometrial cells in endometriotic lesion biopsy samples. Furthermore, we demonstrated that the miRNA profile of peritoneal endometriotic lesion biopsies is largely masked by the surrounding peritoneal tissue, challenging the discovery of an accurate lesion-specific miRNA profile. Taken together, our findings indicate that only particular miRNAs with a significantly higher expression in endometriotic cells can be detected from lesion biopsies, and can serve as diagnostic markers for endometriosis.

Alignment of high-throughput sequencing data inside in-memory databases.

Science.gov (United States)

Firnkorn, Daniel; Knaup-Gregori, Petra; Lorenzo Bermejo, Justo; Ganzinger, Matthias

2014-01-01

In times of high-throughput DNA sequencing techniques, performance-capable analysis of DNA sequences is of high importance. Computer supported DNA analysis is still an intensive time-consuming task. In this paper we explore the potential of a new In-Memory database technology by using SAP's High Performance Analytic Appliance (HANA). We focus on read alignment as one of the first steps in DNA sequence analysis. In particular, we examined the widely used Burrows-Wheeler Aligner (BWA) and implemented stored procedures in both, HANA and the free database system MySQL, to compare execution time and memory management. To ensure that the results are comparable, MySQL has been running in memory as well, utilizing its integrated memory engine for database table creation. We implemented stored procedures, containing exact and inexact searching of DNA reads within the reference genome GRCh37. Due to technical restrictions in SAP HANA concerning recursion, the inexact matching problem could not be implemented on this platform. Hence, performance analysis between HANA and MySQL was made by comparing the execution time of the exact search procedures. Here, HANA was approximately 27 times faster than MySQL which means, that there is a high potential within the new In-Memory concepts, leading to further developments of DNA analysis procedures in the future.

Evolution of blue-flowered species of genus Linum based on high-throughput sequencing of ribosomal RNA genes.

Science.gov (United States)

Bolsheva, Nadezhda L; Melnikova, Nataliya V; Kirov, Ilya V; Speranskaya, Anna S; Krinitsina, Anastasia A; Dmitriev, Alexey A; Belenikin, Maxim S; Krasnov, George S; Lakunina, Valentina A; Snezhkina, Anastasiya V; Rozhmina, Tatiana A; Samatadze, Tatiana E; Yurkevich, Olga Yu; Zoshchuk, Svyatoslav A; Amosova, Аlexandra V; Kudryavtseva, Anna V; Muravenko, Olga V

2017-12-28

The species relationships within the genus Linum have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of Linum still remain unresolved. In particular, the species relationships within the sections Stellerolinum and Dasylinum need further clarification. Also, the question of independence of the species of the section Adenolinum still remains unanswered. Moreover, the relationships of L. narbonense and other species of the section Linum require further clarification. Additionally, the origin of tetraploid species of the section Linum (2n = 30) including the cultivated species L. usitatissimum has not been explored. The present study examines the phylogeny of blue-flowered species of Linum by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes. High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH. Our findings confirm that L. stelleroides forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect. Dasylinum. The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect. Adenolinum. The allotetraploid cultivated species L. usitatissimum and its wild ancestor L. angustifolium (2n = 30) could originate either as the result of hybridization of two diploid species (2n = 16) related to the modern L. gandiflorum and L. decumbens, or hybridization of a diploid species (2n = 16) and a diploid ancestor of modern L. narbonense (2n = 14). High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the

The efficacy of high-throughput sequencing and target enrichment on charred archaeobotanical remains

DEFF Research Database (Denmark)

Nistelberger, H. M.; Smith, O.; Wales, Nathan

2016-01-01

. It has been suggested that high-throughput sequencing (HTS) technologies coupled with DNA enrichment techniques may overcome some of these limitations. Here we report the findings of HTS and target enrichment on four important archaeological crops (barley, grape, maize and rice) performed in three...... lightly-charred maize cob. Even with target enrichment, this sample failed to yield adequate data required to address fundamental questions in archaeology and biology. We further reanalysed part of an existing dataset on charred plant material, and found all purported endogenous DNA sequences were likely...

High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA.

Science.gov (United States)

Kebschull, Justus M; Garcia da Silva, Pedro; Reid, Ashlan P; Peikon, Ian D; Albeanu, Dinu F; Zador, Anthony M

2016-09-07

Neurons transmit information to distant brain regions via long-range axonal projections. In the mouse, area-to-area connections have only been systematically mapped using bulk labeling techniques, which obscure the diverse projections of intermingled single neurons. Here we describe MAPseq (Multiplexed Analysis of Projections by Sequencing), a technique that can map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences ("barcodes"). Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Applying MAPseq to the locus coeruleus (LC), we find that individual LC neurons have preferred cortical targets. By recasting neuroanatomy, which is traditionally viewed as a problem of microscopy, as a problem of sequencing, MAPseq harnesses advances in sequencing technology to permit high-throughput interrogation of brain circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

Using high-throughput sequencing to leverage surveillance of genetic diversity and oseltamivir resistance: a pilot study during the 2009 influenza A(H1N1 pandemic.

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Juan Téllez-Sosa

Full Text Available BACKGROUND: Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The "deep sequencing" approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples. METHODOLOGY AND PRINCIPAL FINDINGS: We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1 pandemic (A(H1N1pdm virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n  =  299 taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July to second wave (September-November of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance. CONCLUSIONS: NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that

HTSeq--a Python framework to work with high-throughput sequencing data.

Science.gov (United States)

Anders, Simon; Pyl, Paul Theodor; Huber, Wolfgang

2015-01-15

A large choice of tools exists for many standard tasks in the analysis of high-throughput sequencing (HTS) data. However, once a project deviates from standard workflows, custom scripts are needed. We present HTSeq, a Python library to facilitate the rapid development of such scripts. HTSeq offers parsers for many common data formats in HTS projects, as well as classes to represent data, such as genomic coordinates, sequences, sequencing reads, alignments, gene model information and variant calls, and provides data structures that allow for querying via genomic coordinates. We also present htseq-count, a tool developed with HTSeq that preprocesses RNA-Seq data for differential expression analysis by counting the overlap of reads with genes. HTSeq is released as an open-source software under the GNU General Public Licence and available from http://www-huber.embl.de/HTSeq or from the Python Package Index at https://pypi.python.org/pypi/HTSeq. © The Author 2014. Published by Oxford University Press.

High Throughput Facility

Data.gov (United States)

Federal Laboratory Consortium — Argonne?s high throughput facility provides highly automated and parallel approaches to material and materials chemistry development. The facility allows scientists...

A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

DEFF Research Database (Denmark)

Uren, Anthony G; Mikkers, Harald; Kool, Jaap

2009-01-01

sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

COMPUTER APPROACHES TO WHEAT HIGH-THROUGHPUT PHENOTYPING

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Afonnikov D.

2012-08-01

Full Text Available The growing need for rapid and accurate approaches for large-scale assessment of phenotypic characters in plants becomes more and more obvious in the studies looking into relationships between genotype and phenotype. This need is due to the advent of high throughput methods for analysis of genomes. Nowadays, any genetic experiment involves data on thousands and dozens of thousands of plants. Traditional ways of assessing most phenotypic characteristics (those with reliance on the eye, the touch, the ruler are little effective on samples of such sizes. Modern approaches seek to take advantage of automated phenotyping, which warrants a much more rapid data acquisition, higher accuracy of the assessment of phenotypic features, measurement of new parameters of these features and exclusion of human subjectivity from the process. Additionally, automation allows measurement data to be rapidly loaded into computer databases, which reduces data processing time.In this work, we present the WheatPGE information system designed to solve the problem of integration of genotypic and phenotypic data and parameters of the environment, as well as to analyze the relationships between the genotype and phenotype in wheat. The system is used to consolidate miscellaneous data on a plant for storing and processing various morphological traits and genotypes of wheat plants as well as data on various environmental factors. The system is available at www.wheatdb.org. Its potential in genetic experiments has been demonstrated in high-throughput phenotyping of wheat leaf pubescence.

Centroid based clustering of high throughput sequencing reads based on n-mer counts.

Science.gov (United States)

Solovyov, Alexander; Lipkin, W Ian

2013-09-08

Many problems in computational biology require alignment-free sequence comparisons. One of the common tasks involving sequence comparison is sequence clustering. Here we apply methods of alignment-free comparison (in particular, comparison using sequence composition) to the challenge of sequence clustering. We study several centroid based algorithms for clustering sequences based on word counts. Study of their performance shows that using k-means algorithm with or without the data whitening is efficient from the computational point of view. A higher clustering accuracy can be achieved using the soft expectation maximization method, whereby each sequence is attributed to each cluster with a specific probability. We implement an open source tool for alignment-free clustering. It is publicly available from github: https://github.com/luscinius/afcluster. We show the utility of alignment-free sequence clustering for high throughput sequencing analysis despite its limitations. In particular, it allows one to perform assembly with reduced resources and a minimal loss of quality. The major factor affecting performance of alignment-free read clustering is the length of the read.

Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing

OpenAIRE

Cui, Li; Zhao, Tingting; Hu, Haibing; Zhang, Wen; Hua, Xiuguo

2017-01-01

Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD) patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by ? diversity and principal coordinate analysis (PCoA). Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA) trees were created. Results. After dat...

High-throughput sequencing of forensic genetic samples using punches of FTA cards with buccal swabs.

Science.gov (United States)

Kampmann, Marie-Louise; Buchard, Anders; Børsting, Claus; Morling, Niels

2016-01-01

Here, we demonstrate that punches from buccal swab samples preserved on FTA cards can be used for high-throughput DNA sequencing, also known as massively parallel sequencing (MPS). We typed 44 reference samples with the HID-Ion AmpliSeq Identity Panel using washed 1.2 mm punches from FTA cards with buccal swabs and compared the results with those obtained with DNA extracted using the EZ1 DNA Investigator Kit. Concordant profiles were obtained for all samples. Our protocol includes simple punch, wash, and PCR steps, reducing cost and hands-on time in the laboratory. Furthermore, it facilitates automation of DNA sequencing.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

Science.gov (United States)

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

Directory of Open Access Journals (Sweden)

Karolina Chwialkowska

2017-11-01

Full Text Available Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq. We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation

Error correction and statistical analyses for intra-host comparisons of feline immunodeficiency virus diversity from high-throughput sequencing data.

Science.gov (United States)

Liu, Yang; Chiaromonte, Francesca; Ross, Howard; Malhotra, Raunaq; Elleder, Daniel; Poss, Mary

2015-06-30

Infection with feline immunodeficiency virus (FIV) causes an immunosuppressive disease whose consequences are less severe if cats are co-infected with an attenuated FIV strain (PLV). We use virus diversity measurements, which reflect replication ability and the virus response to various conditions, to test whether diversity of virulent FIV in lymphoid tissues is altered in the presence of PLV. Our data consisted of the 3' half of the FIV genome from three tissues of animals infected with FIV alone, or with FIV and PLV, sequenced by 454 technology. Since rare variants dominate virus populations, we had to carefully distinguish sequence variation from errors due to experimental protocols and sequencing. We considered an exponential-normal convolution model used for background correction of microarray data, and modified it to formulate an error correction approach for minor allele frequencies derived from high-throughput sequencing. Similar to accounting for over-dispersion in counts, this accounts for error-inflated variability in frequencies - and quite effectively reproduces empirically observed distributions. After obtaining error-corrected minor allele frequencies, we applied ANalysis Of VAriance (ANOVA) based on a linear mixed model and found that conserved sites and transition frequencies in FIV genes differ among tissues of dual and single infected cats. Furthermore, analysis of minor allele frequencies at individual FIV genome sites revealed 242 sites significantly affected by infection status (dual vs. single) or infection status by tissue interaction. All together, our results demonstrated a decrease in FIV diversity in bone marrow in the presence of PLV. Importantly, these effects were weakened or undetectable when error correction was performed with other approaches (thresholding of minor allele frequencies; probabilistic clustering of reads). We also queried the data for cytidine deaminase activity on the viral genome, which causes an asymmetric increase

Leveraging the Power of High Performance Computing for Next Generation Sequencing Data Analysis: Tricks and Twists from a High Throughput Exome Workflow

Science.gov (United States)

Wonczak, Stephan; Thiele, Holger; Nieroda, Lech; Jabbari, Kamel; Borowski, Stefan; Sinha, Vishal; Gunia, Wilfried; Lang, Ulrich; Achter, Viktor; Nürnberg, Peter

2015-01-01

Next generation sequencing (NGS) has been a great success and is now a standard method of research in the life sciences. With this technology, dozens of whole genomes or hundreds of exomes can be sequenced in rather short time, producing huge amounts of data. Complex bioinformatics analyses are required to turn these data into scientific findings. In order to run these analyses fast, automated workflows implemented on high performance computers are state of the art. While providing sufficient compute power and storage to meet the NGS data challenge, high performance computing (HPC) systems require special care when utilized for high throughput processing. This is especially true if the HPC system is shared by different users. Here, stability, robustness and maintainability are as important for automated workflows as speed and throughput. To achieve all of these aims, dedicated solutions have to be developed. In this paper, we present the tricks and twists that we utilized in the implementation of our exome data processing workflow. It may serve as a guideline for other high throughput data analysis projects using a similar infrastructure. The code implementing our solutions is provided in the supporting information files. PMID:25942438

Modeling Disordered Materials with a High Throughput ab-initio Approach

Science.gov (United States)

2015-11-13

Modeling Disordered Materials with a High Throughput ab - initio Approach Kesong Yang,1 Corey Oses,2 and Stefano Curtarolo3, 4 1Department of...J. Furthmüller, Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set, Phys. Rev. B 54, 11169–11186 (1996

High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak

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Trout-Yakel Keri M

2010-02-01

Full Text Available Abstract Background A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE. Results The chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes. Conclusions High-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

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Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

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Greub, Gilbert; Kebbi-Beghdadi, Carole; Bertelli, Claire; Collyn, François; Riederer, Beat M; Yersin, Camille; Croxatto, Antony; Raoult, Didier

2009-12-23

With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

Whole Genome Sequencing of Enterovirus species C Isolates by High-throughput Sequencing: Development of Generic Primers

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Maël Bessaud

2016-08-01

Full Text Available Enteroviruses are among the most common viruses infecting humans and can cause diverse clinical syndromes ranging from minor febrile illness to severe and potentially fatal diseases. Enterovirus species C (EV-C consists of more than 20 types, among which the 3 serotypes of polioviruses, the etiological agents of poliomyelitis, are included. Biodiversity and evolution of EV-C genomes are shaped by frequent recombination events. Therefore, identification and characterization of circulating EV-C strains require the sequencing of different genomic regions.A simple method was developed to sequence quickly the entire genome of EV-C isolates. Four overlapping fragments were produced separately by RT-PCR performed with generic primers. The four amplicons were then pooled and purified prior to be sequenced by high-throughput technique.The method was assessed on a panel of EV-Cs belonging to a wide-range of types. It can be used to determine full-length genome sequences through de novo assembly of thousands of reads. It was also able to discriminate reads from closely related viruses in mixtures.By decreasing the workload compared to classical Sanger-based techniques, this method will serve as a precious tool for sequencing large panels of EV-Cs isolated in cell cultures during environmental surveillance or from patients, including vaccine-derived polioviruses.

HTSstation: a web application and open-access libraries for high-throughput sequencing data analysis.

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David, Fabrice P A; Delafontaine, Julien; Carat, Solenne; Ross, Frederick J; Lefebvre, Gregory; Jarosz, Yohan; Sinclair, Lucas; Noordermeer, Daan; Rougemont, Jacques; Leleu, Marion

2014-01-01

The HTSstation analysis portal is a suite of simple web forms coupled to modular analysis pipelines for various applications of High-Throughput Sequencing including ChIP-seq, RNA-seq, 4C-seq and re-sequencing. HTSstation offers biologists the possibility to rapidly investigate their HTS data using an intuitive web application with heuristically pre-defined parameters. A number of open-source software components have been implemented and can be used to build, configure and run HTS analysis pipelines reactively. Besides, our programming framework empowers developers with the possibility to design their own workflows and integrate additional third-party software. The HTSstation web application is accessible at http://htsstation.epfl.ch.

A CRISPR CASe for High-Throughput Silencing

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Jacob eHeintze

2013-10-01

Full Text Available Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA and zinc finger nuclease technologies. More recently, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated (Cas9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.

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

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

2016-01-01

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

High throughput sequencing and proteomics to identify immunogenic proteins of a new pathogen: the dirty genome approach.

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Gilbert Greub

Full Text Available BACKGROUND: With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive. METHODS/PRINCIPAL FINDINGS: We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA. CONCLUSIONS/SIGNIFICANCE: This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

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Shen Pang

Full Text Available The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

[Study on Microbial Diversity of Peri-implantitis Subgingival by High-throughput Sequencing].

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Li, Zhi-jie; Wang, Shao-guo; Li, Yue-hong; Tu, Dong-xiang; Liu, Shi-yun; Nie, Hong-bing; Li, Zhi-qiang; Zhang, Ju-mei

2015-07-01

To study microbial diversity of peri-implantitis subgingival with high-throughput sequencing, and investigate microbiological etiology of peri-implantitis. Subgingival plaques were sampled from the patients with peri-implantitis (D group) and non-peri-implantitis subjects (N group). The microbiological diversity of the subgingival plaques was detected by sequencing V4 region of 16S rRNA with Illumina Miseq platform. The diversity of the community structure was analyzed using Mothur software. A total of 156 507 gene sequences were detected in nine samples and 4 402 operational taxonomic units (OTUs) were found. Selenomonas, Pseudomonas, and Fusobacterium were dominant bacteria in D group, while Fusobacterium, Veillonella and Streptococcus were dominant bacteria in N group. Differences between peri-implantitis and non-peri-implantitis bacterial communities were observed at all phylogenetic levels by LEfSe, which was also found in PcoA test. The occurrence of peri-implantitis is not only related to periodontitis pathogenic microbe, but also related with the changes of oral microbial community structure. Treponema, Herbaspirillum, Butyricimonas and Phaeobacte may be closely related to the occurrence and development of peri-implantitis.

SNP calling using genotype model selection on high-throughput sequencing data

KAUST Repository

You, Na

2012-01-16

Motivation: A review of the available single nucleotide polymorphism (SNP) calling procedures for Illumina high-throughput sequencing (HTS) platform data reveals that most rely mainly on base-calling and mapping qualities as sources of error when calling SNPs. Thus, errors not involved in base-calling or alignment, such as those in genomic sample preparation, are not accounted for.Results: A novel method of consensus and SNP calling, Genotype Model Selection (GeMS), is given which accounts for the errors that occur during the preparation of the genomic sample. Simulations and real data analyses indicate that GeMS has the best performance balance of sensitivity and positive predictive value among the tested SNP callers. © The Author 2012. Published by Oxford University Press. All rights reserved.

Algorithms for mapping high-throughput DNA sequences

DEFF Research Database (Denmark)

Frellsen, Jes; Menzel, Peter; Krogh, Anders

2014-01-01

of data generation, new bioinformatics approaches have been developed to cope with the large amount of sequencing reads obtained in these experiments. In this chapter, we first introduce HTS technologies and their usage in molecular biology and discuss the problem of mapping sequencing reads...... to their genomic origin. We then in detail describe two approaches that offer very fast heuristics to solve the mapping problem in a feasible runtime. In particular, we describe the BLAT algorithm, and we give an introduction to the Burrows-Wheeler Transform and the mapping algorithms based on this transformation....

Experimental design-based functional mining and characterization of high-throughput sequencing data in the sequence read archive.

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Takeru Nakazato

Full Text Available High-throughput sequencing technology, also called next-generation sequencing (NGS, has the potential to revolutionize the whole process of genome sequencing, transcriptomics, and epigenetics. Sequencing data is captured in a public primary data archive, the Sequence Read Archive (SRA. As of January 2013, data from more than 14,000 projects have been submitted to SRA, which is double that of the previous year. Researchers can download raw sequence data from SRA website to perform further analyses and to compare with their own data. However, it is extremely difficult to search entries and download raw sequences of interests with SRA because the data structure is complicated, and experimental conditions along with raw sequences are partly described in natural language. Additionally, some sequences are of inconsistent quality because anyone can submit sequencing data to SRA with no quality check. Therefore, as a criterion of data quality, we focused on SRA entries that were cited in journal articles. We extracted SRA IDs and PubMed IDs (PMIDs from SRA and full-text versions of journal articles and retrieved 2748 SRA ID-PMID pairs. We constructed a publication list referring to SRA entries. Since, one of the main themes of -omics analyses is clarification of disease mechanisms, we also characterized SRA entries by disease keywords, according to the Medical Subject Headings (MeSH extracted from articles assigned to each SRA entry. We obtained 989 SRA ID-MeSH disease term pairs, and constructed a disease list referring to SRA data. We previously developed feature profiles of diseases in a system called "Gendoo". We generated hyperlinks between diseases extracted from SRA and the feature profiles of it. The developed project, publication and disease lists resulting from this study are available at our web service, called "DBCLS SRA" (http://sra.dbcls.jp/. This service will improve accessibility to high-quality data from SRA.

Scrutinizing virus genome termini by high-throughput sequencing.

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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.

A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection.

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Goodacre, Norman; Aljanahi, Aisha; Nandakumar, Subhiksha; Mikailov, Mike; Khan, Arifa S

2018-01-01

Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have

Application of ToxCast High-Throughput Screening and ...

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Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenesis Distruptors Slide presentation at the SETAC annual meeting on High-Throughput Screening and Modeling Approaches to Identify Steroidogenssis Distruptors

Filtering high-throughput protein-protein interaction data using a combination of genomic features

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Patil Ashwini

2005-04-01

Full Text Available Abstract Background Protein-protein interaction data used in the creation or prediction of molecular networks is usually obtained from large scale or high-throughput experiments. This experimental data is liable to contain a large number of spurious interactions. Hence, there is a need to validate the interactions and filter out the incorrect data before using them in prediction studies. Results In this study, we use a combination of 3 genomic features – structurally known interacting Pfam domains, Gene Ontology annotations and sequence homology – as a means to assign reliability to the protein-protein interactions in Saccharomyces cerevisiae determined by high-throughput experiments. Using Bayesian network approaches, we show that protein-protein interactions from high-throughput data supported by one or more genomic features have a higher likelihood ratio and hence are more likely to be real interactions. Our method has a high sensitivity (90% and good specificity (63%. We show that 56% of the interactions from high-throughput experiments in Saccharomyces cerevisiae have high reliability. We use the method to estimate the number of true interactions in the high-throughput protein-protein interaction data sets in Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens to be 27%, 18% and 68% respectively. Our results are available for searching and downloading at http://helix.protein.osaka-u.ac.jp/htp/. Conclusion A combination of genomic features that include sequence, structure and annotation information is a good predictor of true interactions in large and noisy high-throughput data sets. The method has a very high sensitivity and good specificity and can be used to assign a likelihood ratio, corresponding to the reliability, to each interaction.

The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

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Pietiainen, Vilja; Saarela, Jani; von Schantz, Carina; Turunen, Laura; Ostling, Paivi; Wennerberg, Krister

2014-05-01

The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.

High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

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Weiguo Hou

2018-05-01

Full Text Available Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length encoding the cyanophage gp23 major capsid protein (MCP. Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92% belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

Tracking TCRβ sequence clonotype expansions during antiviral therapy using high-throughput sequencing of the hypervariable region

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Mark W Robinson

2016-04-01

Full Text Available To maintain a persistent infection viruses such as hepatitis C virus (HCV employ a range of mechanisms that subvert protective T cell responses. The suppression of antigen-specific T cell responses by HCV hinders efforts to profile T cell responses during chronic infection and antiviral therapy. Conventional methods of detecting antigen-specific T cells utilise either antigen stimulation (e.g. ELISpot, proliferation assays, cytokine production or antigen-loaded tetramer staining. This limits the ability to profile T cell responses during chronic infection due to suppressed effector function and the requirement for prior knowledge of antigenic viral peptide sequences. Recently high-throughput sequencing (HTS technologies have been developed for the analysis of T cell repertoires. In the present study we have assessed the feasibility of HTS of the TCRβ complementarity determining region (CDR3 to track T cell expansions in an antigen-independent manner. Using sequential blood samples from HCV-infected individuals undergoing anti-viral therapy we were able to measure the population frequencies of >35,000 TCRβ sequence clonotypes in each individual over the course of 12 weeks. TRBV/TRBJ gene segment usage varied markedly between individuals but remained relatively constant within individuals across the course of therapy. Despite this stable TRBV/TRBJ gene segment usage, a number of TCRβ sequence clonotypes showed dramatic changes in read frequency. These changes could not be linked to therapy outcomes in the present study however the TCRβ CDR3 sequences with the largest fold changes did include sequences with identical TRBV/TRBJ gene segment usage and high joining region homology to previously published CDR3 sequences from HCV-specific T cells targeting the HLA-B*0801-restricted 1395HSKKKCDEL1403 and HLA-A*0101–restricted 1435ATDALMTGY1443 epitopes. The pipeline developed in this proof of concept study provides a platform for the design of

Barcoding the food chain: from Sanger to high-throughput sequencing.

Science.gov (United States)

Littlefair, Joanne E; Clare, Elizabeth L

2016-11-01

Society faces the complex challenge of supporting biodiversity and ecosystem functioning, while ensuring food security by providing safe traceable food through an ever-more-complex global food chain. The increase in human mobility brings the added threat of pests, parasites, and invaders that further complicate our agro-industrial efforts. DNA barcoding technologies allow researchers to identify both individual species, and, when combined with universal primers and high-throughput sequencing techniques, the diversity within mixed samples (metabarcoding). These tools are already being employed to detect market substitutions, trace pests through the forensic evaluation of trace "environmental DNA", and to track parasitic infections in livestock. The potential of DNA barcoding to contribute to increased security of the food chain is clear, but challenges remain in regulation and the need for validation of experimental analysis. Here, we present an overview of the current uses and challenges of applied DNA barcoding in agriculture, from agro-ecosystems within farmland to the kitchen table.

Digital PCR provides sensitive and absolute calibration for high throughput sequencing

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Fan H Christina

2009-03-01

Full Text Available Abstract Background Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing. Results We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth. Conclusion The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.

Ultraspecific probes for high throughput HLA typing

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Eggers Rick

2009-02-01

Full Text Available Abstract Background The variations within an individual's HLA (Human Leukocyte Antigen genes have been linked to many immunological events, e.g. susceptibility to disease, response to vaccines, and the success of blood, tissue, and organ transplants. Although the microarray format has the potential to achieve high-resolution typing, this has yet to be attained due to inefficiencies of current probe design strategies. Results We present a novel three-step approach for the design of high-throughput microarray assays for HLA typing. This approach first selects sequences containing the SNPs present in all alleles of the locus of interest and next calculates the number of base changes necessary to convert a candidate probe sequences to the closest subsequence within the set of sequences that are likely to be present in the sample including the remainder of the human genome in order to identify those candidate probes which are "ultraspecific" for the allele of interest. Due to the high specificity of these sequences, it is possible that preliminary steps such as PCR amplification are no longer necessary. Lastly, the minimum number of these ultraspecific probes is selected such that the highest resolution typing can be achieved for the minimal cost of production. As an example, an array was designed and in silico results were obtained for typing of the HLA-B locus. Conclusion The assay presented here provides a higher resolution than has previously been developed and includes more alleles than previously considered. Based upon the in silico and preliminary experimental results, we believe that the proposed approach can be readily applied to any highly polymorphic gene system.

SEED 2: a user-friendly platform for amplicon high-throughput sequencing data analyses.

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Vetrovský, Tomáš; Baldrian, Petr; Morais, Daniel; Berger, Bonnie

2018-02-14

Modern molecular methods have increased our ability to describe microbial communities. Along with the advances brought by new sequencing technologies, we now require intensive computational resources to make sense of the large numbers of sequences continuously produced. The software developed by the scientific community to address this demand, although very useful, require experience of the command-line environment, extensive training and have steep learning curves, limiting their use. We created SEED 2, a graphical user interface for handling high-throughput amplicon-sequencing data under Windows operating systems. SEED 2 is the only sequence visualizer that empowers users with tools to handle amplicon-sequencing data of microbial community markers. It is suitable for any marker genes sequences obtained through Illumina, IonTorrent or Sanger sequencing. SEED 2 allows the user to process raw sequencing data, identify specific taxa, produce of OTU-tables, create sequence alignments and construct phylogenetic trees. Standard dual core laptops with 8 GB of RAM can handle ca. 8 million of Illumina PE 300 bp sequences, ca. 4GB of data. SEED 2 was implemented in Object Pascal and uses internal functions and external software for amplicon data processing. SEED 2 is a freeware software, available at http://www.biomed.cas.cz/mbu/lbwrf/seed/ as a self-contained file, including all the dependencies, and does not require installation. Supplementary data contain a comprehensive list of supported functions. daniel.morais@biomed.cas.cz. Supplementary data are available at Bioinformatics online. © The Author(s) 2018. Published by Oxford University Press.

Enhanced throughput for infrared automated DNA sequencing

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Middendorf, Lyle R.; Gartside, Bill O.; Humphrey, Pat G.; Roemer, Stephen C.; Sorensen, David R.; Steffens, David L.; Sutter, Scott L.

1995-04-01

Several enhancements have been developed and applied to infrared automated DNA sequencing resulting in significantly higher throughput. A 41 cm sequencing gel (31 cm well- to-read distance) combines high resolution of DNA sequencing fragments with optimized run times yielding two runs per day of 500 bases per sample. A 66 cm sequencing gel (56 cm well-to-read distance) produces sequence read lengths of up to 1000 bases for ds and ss templates using either T7 polymerase or cycle-sequencing protocols. Using a multichannel syringe to load 64 lanes allows 16 samples (compatible with 96-well format) to be visualized for each run. The 41 cm gel configuration allows 16,000 bases per day (16 samples X 500 bases/sample X 2 ten hour runs/day) to be sequenced with the advantages of infrared technology. Enhancements to internal labeling techniques using an infrared-labeled dATP molecule (Boehringer Mannheim GmbH, Penzberg, Germany; Sequenase (U.S. Biochemical) have also been made. The inclusion of glycerol in the sequencing reactions yields greatly improved results for some primer and template combinations. The inclusion of (alpha) -Thio-dNTP's in the labeling reaction increases signal intensity two- to three-fold.

A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites.

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Côté, Nathalie M L; Daligault, Julien; Pruvost, Mélanie; Bennett, E Andrew; Gorgé, Olivier; Guimaraes, Silvia; Capelli, Nicolas; Le Bailly, Matthieu; Geigl, Eva-Maria; Grange, Thierry

2016-01-01

Human gastrointestinal parasites are good indicators for hygienic conditions and health status of past and present individuals and communities. While microscopic analysis of eggs in sediments of archeological sites often allows their taxonomic identification, this method is rarely effective at the species level, and requires both the survival of intact eggs and their proper identification. Genotyping via PCR-based approaches has the potential to achieve a precise species-level taxonomic determination. However, so far it has mostly been applied to individual eggs isolated from archeological samples. To increase the throughput and taxonomic accuracy, as well as reduce costs of genotyping methods, we adapted a PCR-based approach coupled with next-generation sequencing to perform precise taxonomic identification of parasitic helminths directly from archeological sediments. Our study of twenty-five 100 to 7,200 year-old archeological samples proved this to be a powerful, reliable and efficient approach for species determination even in the absence of preserved eggs, either as a stand-alone method or as a complement to microscopic studies.

Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey

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Varala Kranthi

2007-05-01

Full Text Available Abstract Background Extensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA. Results We sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis. Conclusion This approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.

Characterization of the indigenous microflora in raw and pasteurized buffalo milk during storage at refrigeration temperature by high-throughput sequencing

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The effect of refrigeration on bacterial communities within raw and pasteurized buffalo milk was studied using high-throughput sequencing. High quality samples of raw buffalo milk were obtained from five dairy farms in the Guangxi province of China. A sample of each milk was pasteurized, and both r...

Pyicos: a versatile toolkit for the analysis of high-throughput sequencing data.

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Althammer, Sonja; González-Vallinas, Juan; Ballaré, Cecilia; Beato, Miguel; Eyras, Eduardo

2011-12-15

High-throughput sequencing (HTS) has revolutionized gene regulation studies and is now fundamental for the detection of protein-DNA and protein-RNA binding, as well as for measuring RNA expression. With increasing variety and sequencing depth of HTS datasets, the need for more flexible and memory-efficient tools to analyse them is growing. We describe Pyicos, a powerful toolkit for the analysis of mapped reads from diverse HTS experiments: ChIP-Seq, either punctuated or broad signals, CLIP-Seq and RNA-Seq. We prove the effectiveness of Pyicos to select for significant signals and show that its accuracy is comparable and sometimes superior to that of methods specifically designed for each particular type of experiment. Pyicos facilitates the analysis of a variety of HTS datatypes through its flexibility and memory efficiency, providing a useful framework for data integration into models of regulatory genomics. Open-source software, with tutorials and protocol files, is available at http://regulatorygenomics.upf.edu/pyicos or as a Galaxy server at http://regulatorygenomics.upf.edu/galaxy eduardo.eyras@upf.edu Supplementary data are available at Bioinformatics online.

Bacterial Pathogens and Community Composition in Advanced Sewage Treatment Systems Revealed by Metagenomics Analysis Based on High-Throughput Sequencing

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Lu, Xin; Zhang, Xu-Xiang; Wang, Zhu; Huang, Kailong; Wang, Yuan; Liang, Weigang; Tan, Yunfei; Liu, Bo; Tang, Junying

2015-01-01

This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence. PMID:25938416

BOOGIE: Predicting Blood Groups from High Throughput Sequencing Data.

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Giollo, Manuel; Minervini, Giovanni; Scalzotto, Marta; Leonardi, Emanuela; Ferrari, Carlo; Tosatto, Silvio C E

2015-01-01

Over the last decade, we have witnessed an incredible growth in the amount of available genotype data due to high throughput sequencing (HTS) techniques. This information may be used to predict phenotypes of medical relevance, and pave the way towards personalized medicine. Blood phenotypes (e.g. ABO and Rh) are a purely genetic trait that has been extensively studied for decades, with currently over thirty known blood groups. Given the public availability of blood group data, it is of interest to predict these phenotypes from HTS data which may translate into more accurate blood typing in clinical practice. Here we propose BOOGIE, a fast predictor for the inference of blood groups from single nucleotide variant (SNV) databases. We focus on the prediction of thirty blood groups ranging from the well known ABO and Rh, to the less studied Junior or Diego. BOOGIE correctly predicted the blood group with 94% accuracy for the Personal Genome Project whole genome profiles where good quality SNV annotation was available. Additionally, our tool produces a high quality haplotype phase, which is of interest in the context of ethnicity-specific polymorphisms or traits. The versatility and simplicity of the analysis make it easily interpretable and allow easy extension of the protocol towards other phenotypes. BOOGIE can be downloaded from URL http://protein.bio.unipd.it/download/.

Transcriptomic analysis of Petunia hybrida in response to salt stress using high throughput RNA sequencing.

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Gonzalo H Villarino

Full Text Available Salinity and drought stress are the primary cause of crop losses worldwide. In sodic saline soils sodium chloride (NaCl disrupts normal plant growth and development. The complex interactions of plant systems with abiotic stress have made RNA sequencing a more holistic and appealing approach to study transcriptome level responses in a single cell and/or tissue. In this work, we determined the Petunia transcriptome response to NaCl stress by sequencing leaf samples and assembling 196 million Illumina reads with Trinity software. Using our reference transcriptome we identified more than 7,000 genes that were differentially expressed within 24 h of acute NaCl stress. The proposed transcriptome can also be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome and it is available at the SOL Genomics Network (SGN http://solgenomics.net. Genes related to regulation of reactive oxygen species, transport, and signal transductions as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. The candidate genes identified in this study can be applied as markers for breeding or to genetically engineer plants to enhance salt tolerance. Gene Ontology analyses indicated that most of the NaCl damage happened at 24 h inducing genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. Finally, we report a modification to the library preparation protocol whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. The methodological improvement presented here could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments.

Transcriptomic analysis of Petunia hybrida in response to salt stress using high throughput RNA sequencing.

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Villarino, Gonzalo H; Bombarely, Aureliano; Giovannoni, James J; Scanlon, Michael J; Mattson, Neil S

2014-01-01

Salinity and drought stress are the primary cause of crop losses worldwide. In sodic saline soils sodium chloride (NaCl) disrupts normal plant growth and development. The complex interactions of plant systems with abiotic stress have made RNA sequencing a more holistic and appealing approach to study transcriptome level responses in a single cell and/or tissue. In this work, we determined the Petunia transcriptome response to NaCl stress by sequencing leaf samples and assembling 196 million Illumina reads with Trinity software. Using our reference transcriptome we identified more than 7,000 genes that were differentially expressed within 24 h of acute NaCl stress. The proposed transcriptome can also be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome and it is available at the SOL Genomics Network (SGN) http://solgenomics.net. Genes related to regulation of reactive oxygen species, transport, and signal transductions as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. The candidate genes identified in this study can be applied as markers for breeding or to genetically engineer plants to enhance salt tolerance. Gene Ontology analyses indicated that most of the NaCl damage happened at 24 h inducing genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. Finally, we report a modification to the library preparation protocol whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. The methodological improvement presented here could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments.

A high-throughput multiplex method adapted for GMO detection.

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Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

2008-12-24

A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

High-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination.

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Tan, Yann-Chong; Blum, Lisa K; Kongpachith, Sarah; Ju, Chia-Hsin; Cai, Xiaoyong; Lindstrom, Tamsin M; Sokolove, Jeremy; Robinson, William H

2014-03-01

We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination. Published by Elsevier Inc.

Metabolomic and high-throughput sequencing analysis—modern approach for the assessment of biodeterioration of materials from historic buildings

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Gutarowska, Beata; Celikkol-Aydin, Sukriye; Bonifay, Vincent; Otlewska, Anna; Aydin, Egemen; Oldham, Athenia L.; Brauer, Jonathan I.; Duncan, Kathleen E.; Adamiak, Justyna; Sunner, Jan A.; Beech, Iwona B.

2015-01-01

Preservation of cultural heritage is of paramount importance worldwide. Microbial colonization of construction materials, such as wood, brick, mortar, and stone in historic buildings can lead to severe deterioration. The aim of the present study was to give modern insight into the phylogenetic diversity and activated metabolic pathways of microbial communities colonized historic objects located in the former Auschwitz II–Birkenau concentration and extermination camp in Oświecim, Poland. For this purpose we combined molecular, microscopic and chemical methods. Selected specimens were examined using Field Emission Scanning Electron Microscopy (FESEM), metabolomic analysis and high-throughput Illumina sequencing. FESEM imaging revealed the presence of complex microbial communities comprising diatoms, fungi and bacteria, mainly cyanobacteria and actinobacteria, on sample surfaces. Microbial diversity of brick specimens appeared higher than that of the wood and was dominated by algae and cyanobacteria, while wood was mainly colonized by fungi. DNA sequences documented the presence of 15 bacterial phyla representing 99 genera including Halomonas, Halorhodospira, Salinisphaera, Salinibacterium, Rubrobacter, Streptomyces, Arthrobacter and nine fungal classes represented by 113 genera including Cladosporium, Acremonium, Alternaria, Engyodontium, Penicillium, Rhizopus, and Aureobasidium. Most of the identified sequences were characteristic of organisms implicated in deterioration of wood and brick. Metabolomic data indicated the activation of numerous metabolic pathways, including those regulating the production of primary and secondary metabolites, for example, metabolites associated with the production of antibiotics, organic acids and deterioration of organic compounds. The study demonstrated that a combination of electron microscopy imaging with metabolomic and genomic techniques allows to link the phylogenetic information and metabolic profiles of microbial

Metabolomic and high-throughput sequencing analysis-modern approach for the assessment of biodeterioration of materials from historic buildings.

Science.gov (United States)

Gutarowska, Beata; Celikkol-Aydin, Sukriye; Bonifay, Vincent; Otlewska, Anna; Aydin, Egemen; Oldham, Athenia L; Brauer, Jonathan I; Duncan, Kathleen E; Adamiak, Justyna; Sunner, Jan A; Beech, Iwona B

2015-01-01

Preservation of cultural heritage is of paramount importance worldwide. Microbial colonization of construction materials, such as wood, brick, mortar, and stone in historic buildings can lead to severe deterioration. The aim of the present study was to give modern insight into the phylogenetic diversity and activated metabolic pathways of microbial communities colonized historic objects located in the former Auschwitz II-Birkenau concentration and extermination camp in Oświecim, Poland. For this purpose we combined molecular, microscopic and chemical methods. Selected specimens were examined using Field Emission Scanning Electron Microscopy (FESEM), metabolomic analysis and high-throughput Illumina sequencing. FESEM imaging revealed the presence of complex microbial communities comprising diatoms, fungi and bacteria, mainly cyanobacteria and actinobacteria, on sample surfaces. Microbial diversity of brick specimens appeared higher than that of the wood and was dominated by algae and cyanobacteria, while wood was mainly colonized by fungi. DNA sequences documented the presence of 15 bacterial phyla representing 99 genera including Halomonas, Halorhodospira, Salinisphaera, Salinibacterium, Rubrobacter, Streptomyces, Arthrobacter and nine fungal classes represented by 113 genera including Cladosporium, Acremonium, Alternaria, Engyodontium, Penicillium, Rhizopus, and Aureobasidium. Most of the identified sequences were characteristic of organisms implicated in deterioration of wood and brick. Metabolomic data indicated the activation of numerous metabolic pathways, including those regulating the production of primary and secondary metabolites, for example, metabolites associated with the production of antibiotics, organic acids and deterioration of organic compounds. The study demonstrated that a combination of electron microscopy imaging with metabolomic and genomic techniques allows to link the phylogenetic information and metabolic profiles of microbial communities

Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa

NARCIS (Netherlands)

Geisen, S.; Laros, I.; Vizcaino, A.; Bonkowski, M.; Groot, de G.A.

2015-01-01

Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into

High throughput protein production screening

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Beernink, Peter T [Walnut Creek, CA; Coleman, Matthew A [Oakland, CA; Segelke, Brent W [San Ramon, CA

2009-09-08

Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

New approach for high-throughput screening of drug activity on Plasmodium liver stages.

NARCIS (Netherlands)

Gego, A.; Silvie, O.; Franetich, J.F.; Farhati, K.; Hannoun, L.; Luty, A.J.F.; Sauerwein, R.W.; Boucheix, C.; Rubinstein, E.; Mazier, D.

2006-01-01

Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an

Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

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Mendoza, Lucía M; Neef, Alexander; Vignolo, Graciela; Belloch, Carmela

2017-10-01

Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae.

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Jared W Wenger

2010-05-01

Full Text Available Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

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Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

High-Throughput Sequencing, a VersatileWeapon to Support Genome-Based Diagnosis in Infectious Diseases: Applications to Clinical Bacteriology

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Ségolène Caboche

2014-04-01

Full Text Available The recent progresses of high-throughput sequencing (HTS technologies enable easy and cost-reduced access to whole genome sequencing (WGS or re-sequencing. HTS associated with adapted, automatic and fast bioinformatics solutions for sequencing applications promises an accurate and timely identification and characterization of pathogenic agents. Many studies have demonstrated that data obtained from HTS analysis have allowed genome-based diagnosis, which has been consistent with phenotypic observations. These proofs of concept are probably the first steps toward the future of clinical microbiology. From concept to routine use, many parameters need to be considered to promote HTS as a powerful tool to help physicians and clinicians in microbiological investigations. This review highlights the milestones to be completed toward this purpose.

Biphasic Study to Characterize Agricultural Biogas Plants by High-Throughput 16S rRNA Gene Amplicon Sequencing and Microscopic Analysis.

Science.gov (United States)

Maus, Irena; Kim, Yong Sung; Wibberg, Daniel; Stolze, Yvonne; Off, Sandra; Antonczyk, Sebastian; Pühler, Alfred; Scherer, Paul; Schlüter, Andreas

2017-02-28

Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus , were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.

Towards high-throughput phenotyping of complex patterned behaviors in rodents: focus on mouse self-grooming and its sequencing.

Science.gov (United States)

Kyzar, Evan; Gaikwad, Siddharth; Roth, Andrew; Green, Jeremy; Pham, Mimi; Stewart, Adam; Liang, Yiqing; Kobla, Vikrant; Kalueff, Allan V

2011-12-01

Increasingly recognized in biological psychiatry, rodent self-grooming is a complex patterned behavior with evolutionarily conserved cephalo-caudal progression. While grooming is traditionally assessed by the latency, frequency and duration, its sequencing represents another important domain sensitive to various experimental manipulations. Such behavioral complexity requires novel objective approaches to quantify rodent grooming, in addition to time-consuming and highly variable manual observation. The present study combined modern behavior-recognition video-tracking technologies (CleverSys, Inc.) with manual observation to characterize in-depth spontaneous (novelty-induced) and artificial (water-induced) self-grooming in adult male C57BL/6J mice. We specifically focused on individual episodes of grooming (paw licking, head washing, body/leg washing, and tail/genital grooming), their duration and transitions between episodes. Overall, the frequency, duration and transitions detected using the automated approach significantly correlated with manual observations (R=0.51-0.7, pgrooming, also indicating that behavior-recognition tools can be applied to characterize both the amount and sequential organization (patterning) of rodent grooming. Together with further refinement and methodological advancement, this approach will foster high-throughput neurophenotyping of grooming, with multiple applications in drug screening and testing of genetically modified animals. Copyright © 2011 Elsevier B.V. All rights reserved.

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

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

glbase: a framework for combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing data

Directory of Open Access Journals (Sweden)

Andrew Paul Hutchins

2014-01-01

Full Text Available Genomic datasets and the tools to analyze them have proliferated at an astonishing rate. However, such tools are often poorly integrated with each other: each program typically produces its own custom output in a variety of non-standard file formats. Here we present glbase, a framework that uses a flexible set of descriptors that can quickly parse non-binary data files. glbase includes many functions to intersect two lists of data, including operations on genomic interval data and support for the efficient random access to huge genomic data files. Many glbase functions can produce graphical outputs, including scatter plots, heatmaps, boxplots and other common analytical displays of high-throughput data such as RNA-seq, ChIP-seq and microarray expression data. glbase is designed to rapidly bring biological data into a Python-based analytical environment to facilitate analysis and data processing. In summary, glbase is a flexible and multifunctional toolkit that allows the combination and analysis of high-throughput data (especially next-generation sequencing and genome-wide data, and which has been instrumental in the analysis of complex data sets. glbase is freely available at http://bitbucket.org/oaxiom/glbase/.

glbase: a framework for combining, analyzing and displaying heterogeneous genomic and high-throughput sequencing data.

Science.gov (United States)

Hutchins, Andrew Paul; Jauch, Ralf; Dyla, Mateusz; Miranda-Saavedra, Diego

2014-01-01

Genomic datasets and the tools to analyze them have proliferated at an astonishing rate. However, such tools are often poorly integrated with each other: each program typically produces its own custom output in a variety of non-standard file formats. Here we present glbase, a framework that uses a flexible set of descriptors that can quickly parse non-binary data files. glbase includes many functions to intersect two lists of data, including operations on genomic interval data and support for the efficient random access to huge genomic data files. Many glbase functions can produce graphical outputs, including scatter plots, heatmaps, boxplots and other common analytical displays of high-throughput data such as RNA-seq, ChIP-seq and microarray expression data. glbase is designed to rapidly bring biological data into a Python-based analytical environment to facilitate analysis and data processing. In summary, glbase is a flexible and multifunctional toolkit that allows the combination and analysis of high-throughput data (especially next-generation sequencing and genome-wide data), and which has been instrumental in the analysis of complex data sets. glbase is freely available at http://bitbucket.org/oaxiom/glbase/.

Metagenomic analysis and functional characterization of the biogas microbiome using high throughput shotgun sequencing and a novel binning strategy

DEFF Research Database (Denmark)

Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis

2016-01-01

Biogas production is an economically attractive technology that has gained momentum worldwide over the past years. Biogas is produced by a biologically mediated process, widely known as "anaerobic digestion." This process is performed by a specialized and complex microbial community, in which...... performed using >400 proteins revealed that the biogas community is a trove of new species. A new approach based on functional properties as per network representation was developed to assign roles to the microbial species. The organization of the anaerobic digestion microbiome is resembled by a funnel...... on the phylogenetic and functional characterization of the microbial community populating biogas reactors. By applying for the first time high-throughput sequencing and a novel binning strategy, the identified genes were anchored to single genomes providing a clear understanding of their metabolic pathways...

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

Science.gov (United States)

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Morphology control in polymer blend fibers—a high throughput computing approach

Science.gov (United States)

Sesha Sarath Pokuri, Balaji; Ganapathysubramanian, Baskar

2016-08-01

Fibers made from polymer blends have conventionally enjoyed wide use, particularly in textiles. This wide applicability is primarily aided by the ease of manufacturing such fibers. More recently, the ability to tailor the internal morphology of polymer blend fibers by carefully designing processing conditions has enabled such fibers to be used in technologically relevant applications. Some examples include anisotropic insulating properties for heat and anisotropic wicking of moisture, coaxial morphologies for optical applications as well as fibers with high internal surface area for filtration and catalysis applications. However, identifying the appropriate processing conditions from the large space of possibilities using conventional trial-and-error approaches is a tedious and resource-intensive process. Here, we illustrate a high throughput computational approach to rapidly explore and characterize how processing conditions (specifically blend ratio and evaporation rates) affect the internal morphology of polymer blends during solvent based fabrication. We focus on a PS: PMMA system and identify two distinct classes of morphologies formed due to variations in the processing conditions. We subsequently map the processing conditions to the morphology class, thus constructing a ‘phase diagram’ that enables rapid identification of processing parameters for specific morphology class. We finally demonstrate the potential for time dependent processing conditions to get desired features of the morphology. This opens up the possibility of rational stage-wise design of processing pathways for tailored fiber morphology using high throughput computing.

Metabolomic and high-throughput sequencing analysis – modern approach for the assessment of biodeterioration of materials from historic buildings

Directory of Open Access Journals (Sweden)

Beata eGutarowska

2015-09-01

Full Text Available Preservation of cultural heritage is of paramount importance worldwide. Microbial colonization of construction materials, such as wood, brick, mortar and stone in historic buildings can lead to severe deterioration. The aim of the present study was to give modern insight into the phylogenetic diversity and activated metabolic pathways of microbial communities colonized historic objects located in the former Auschwitz II-Birkenau concentration and extermination camp in Oświęcim, Poland. For this purpose we combined molecular, microscopic and chemical methods. Selected specimens were examined using Field Emission Scanning Electron Microscopy (FESEM, metabolomic analysis and high-throughput Illumina sequencing. FESEM imaging revealed the presence of complex microbial communities comprising diatoms, fungi and bacteria, mainly cyanobacteria and actinobacteria, on sample surfaces. Microbial diversity of brick specimens appeared higher than that of the wood and was dominated by algae and cyanobacteria, while wood was mainly colonized by fungi. DNA sequences documented the presence of 15 bacterial phyla representing 99 genera including Halomonas, Halorhodospira, Salinisphaera, Salinibacterium, Rubrobacter, Streptomyces, Arthrobacter and 9 fungal classes represented by 113 genera including Cladosporium, Acremonium, Alternaria, Engyodontium, Penicillium, Rhizopus and Aureobasidium. Most of the identified sequences were characteristic of organisms implicated in deterioration of wood and brick. Metabolomic data indicated the activation of numerous metabolic pathways, including those regulating the production of primary and secondary metabolites, for example, metabolites associated with the production of antibiotics, organic acids and deterioration of organic compounds. The study demonstrated that a combination of electron microscopy imaging with metabolomic and genomic techniques allows to link the phylogenetic information and metabolic profiles of

Metagenomic analysis and functional characterization of the biogas microbiome using high throughput shotgun sequencing and a novel binning strategy.

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Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis G; De Francisci, Davide; Valle, Giorgio; Angelidaki, Irini

2016-01-01

Biogas production is an economically attractive technology that has gained momentum worldwide over the past years. Biogas is produced by a biologically mediated process, widely known as "anaerobic digestion." This process is performed by a specialized and complex microbial community, in which different members have distinct roles in the establishment of a collective organization. Deciphering the complex microbial community engaged in this process is interesting both for unraveling the network of bacterial interactions and for applicability potential to the derived knowledge. In this study, we dissect the bioma involved in anaerobic digestion by means of high throughput Illumina sequencing (~51 gigabases of sequence data), disclosing nearly one million genes and extracting 106 microbial genomes by a novel strategy combining two binning processes. Microbial phylogeny and putative taxonomy performed using >400 proteins revealed that the biogas community is a trove of new species. A new approach based on functional properties as per network representation was developed to assign roles to the microbial species. The organization of the anaerobic digestion microbiome is resembled by a funnel concept, in which the microbial consortium presents a progressive functional specialization while reaching the final step of the process (i.e., methanogenesis). Key microbial genomes encoding enzymes involved in specific metabolic pathways, such as carbohydrates utilization, fatty acids degradation, amino acids fermentation, and syntrophic acetate oxidation, were identified. Additionally, the analysis identified a new uncultured archaeon that was putatively related to Methanomassiliicoccales but surprisingly having a methylotrophic methanogenic pathway. This study is a pioneer research on the phylogenetic and functional characterization of the microbial community populating biogas reactors. By applying for the first time high-throughput sequencing and a novel binning strategy, the

High throughput sequencing identifies chilling responsive genes in sweetpotato (Ipomoea batatas Lam.) during storage.

Science.gov (United States)

Xie, Zeyi; Zhou, Zhilin; Li, Hongmin; Yu, Jingjing; Jiang, Jiaojiao; Tang, Zhonghou; Ma, Daifu; Zhang, Baohong; Han, Yonghua; Li, Zongyun

2018-05-21

Sweetpotato (Ipomoea batatas L.) is a globally important economic food crop. It belongs to Convolvulaceae family and origins in the tropics; however, sweetpotato is sensitive to cold stress during storage. In this study, we performed transcriptome sequencing to investigate the sweetpotato response to chilling stress during storage. A total of 110,110 unigenes were generated via high-throughput sequencing. Differentially expressed genes (DEGs) analysis showed that 18,681 genes were up-regulated and 21,983 genes were down-regulated in low temperature condition. Many DEGs were related to the cell membrane system, antioxidant enzymes, carbohydrate metabolism, and hormone metabolism, which are potentially associated with sweetpotato resistance to low temperature. The existence of DEGs suggests a molecular basis for the biochemical and physiological consequences of sweetpotato in low temperature storage conditions. Our analysis will provide a new target for enhancement of sweetpotato cold stress tolerance in postharvest storage through genetic manipulation. Copyright © 2018. Published by Elsevier Inc.

Data from: Not all are free-living: high-throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa

NARCIS (Netherlands)

Geisen, Stefan; Laros, I.; Vizcaino, A.; Bonkowski, M.; Groot, de G.A.

2015-01-01

Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into

Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

Science.gov (United States)

Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

2016-04-15

In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

SSR_pipeline--computer software for the identification of microsatellite sequences from paired-end Illumina high-throughput DNA sequence data

Science.gov (United States)

Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (SSRs; for example, microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains three analysis modules along with a fourth control module that can be used to automate analyses of large volumes of data. The modules are used to (1) identify the subset of paired-end sequences that pass quality standards, (2) align paired-end reads into a single composite DNA sequence, and (3) identify sequences that possess microsatellites conforming to user specified parameters. Each of the three separate analysis modules also can be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc). All modules are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, Windows). The program suite relies on a compiled Python extension module to perform paired-end alignments. Instructions for compiling the extension from source code are provided in the documentation. Users who do not have Python installed on their computers or who do not have the ability to compile software also may choose to download packaged executable files. These files include all Python scripts, a copy of the compiled extension module, and a minimal installation of Python in a single binary executable. See program documentation for more information.

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology

DEFF Research Database (Denmark)

Nag, Sidsel; Dalgaard, Marlene Danner; Kofoed, Poul-Erik

2017-01-01

Genetic polymorphisms in P. falciparum can be used to indicate the parasite's susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom...... designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well...

High-throughput sequencing, characterization and detection of new and conserved cucumber miRNAs.

Directory of Open Access Journals (Sweden)

Germán Martínez

Full Text Available Micro RNAS (miRNAs are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance. Here we report a combined approach of bioinformatics prediction, high-throughput sequencing data and molecular methods to analyze miRNAs populations in cucumber (Cucumis sativus plants. A set of 19 conserved and 6 known but non-conserved miRNA families were found in our cucumber small RNA dataset. We also identified 7 (3 with their miRNA* strand not previously described miRNAs, candidates to be cucumber-specific. To validate their description these new C. sativus miRNAs were detected by northern blot hybridization. Additionally, potential targets for most conserved and new miRNAs were identified in cucumber genome.In summary, in this study we have identified, by first time, conserved, known non-conserved and new miRNAs arising from an agronomically important species such as C. sativus. The detection of this complex population of regulatory small RNAs suggests that similarly to that observe in other plant species, cucumber miRNAs may possibly play an important role in diverse biological and metabolic processes.

Spatially conserved regulatory elements identified within human and mouse Cd247 gene using high-throughput sequencing data from the ENCODE project

DEFF Research Database (Denmark)

Pundhir, Sachin; Hannibal, Tine Dahlbæk; Bang-Berthelsen, Claus Heiner

2014-01-01

. In this study, we have utilized the wealth of high-throughput sequencing data produced during the Encyclopedia of DNA Elements (ENCODE) project to identify spatially conserved regulatory elements within the Cd247 gene from human and mouse. We show the presence of two transcription factor binding sites...

Sequence protein identification by randomized sequence database and transcriptome mass spectrometry (SPIDER-TMS): from manual to automatic application of a 'de novo sequencing' approach.

Science.gov (United States)

Pascale, Raffaella; Grossi, Gerarda; Cruciani, Gabriele; Mecca, Giansalvatore; Santoro, Donatello; Sarli Calace, Renzo; Falabella, Patrizia; Bianco, Giuliana

Sequence protein identification by a randomized sequence database and transcriptome mass spectrometry software package has been developed at the University of Basilicata in Potenza (Italy) and designed to facilitate the determination of the amino acid sequence of a peptide as well as an unequivocal identification of proteins in a high-throughput manner with enormous advantages of time, economical resource and expertise. The software package is a valid tool for the automation of a de novo sequencing approach, overcoming the main limits and a versatile platform useful in the proteomic field for an unequivocal identification of proteins, starting from tandem mass spectrometry data. The strength of this software is that it is a user-friendly and non-statistical approach, so protein identification can be considered unambiguous.

The simple fool's guide to population genomics via RNA-Seq: An introduction to high-throughput sequencing data analysis

DEFF Research Database (Denmark)

De Wit, P.; Pespeni, M.H.; Ladner, J.T.

2012-01-01

to Population Genomics via RNA-seq' (SFG), a document intended to serve as an easy-to-follow protocol, walking a user through one example of high-throughput sequencing data analysis of nonmodel organisms. It is by no means an exhaustive protocol, but rather serves as an introduction to the bioinformatic methods...... used in population genomics, enabling a user to gain familiarity with basic analysis steps. The SFG consists of two parts. This document summarizes the steps needed and lays out the basic themes for each and a simple approach to follow. The second document is the full SFG, publicly available at http://sfg.......stanford.edu, that includes detailed protocols for data processing and analysis, along with a repository of custom-made scripts and sample files. Steps included in the SFG range from tissue collection to de novo assembly, blast annotation, alignment, gene expression, functional enrichment, SNP detection, principal components...

High-throughput sequencing and mutagenesis to accelerate the domestication of Microlaena stipoides as a new food crop.

Directory of Open Access Journals (Sweden)

Frances M Shapter

Full Text Available Global food demand, climatic variability and reduced land availability are driving the need for domestication of new crop species. The accelerated domestication of a rice-like Australian dryland polyploid grass, Microlaena stipoides (Poaceae, was targeted using chemical mutagenesis in conjunction with high throughput sequencing of genes for key domestication traits. While M. stipoides has previously been identified as having potential as a new grain crop for human consumption, only a limited understanding of its genetic diversity and breeding system was available to aid the domestication process. Next generation sequencing of deeply-pooled target amplicons estimated allelic diversity of a selected base population at 14.3 SNP/Mb and identified novel, putatively mutation-induced polymorphisms at about 2.4 mutations/Mb. A 97% lethal dose (LD₉₇ of ethyl methanesulfonate treatment was applied without inducing sterility in this polyploid species. Forward and reverse genetic screens identified beneficial alleles for the domestication trait, seed-shattering. Unique phenotypes observed in the M2 population suggest the potential for rapid accumulation of beneficial traits without recourse to a traditional cross-breeding strategy. This approach may be applicable to other wild species, unlocking their potential as new food, fibre and fuel crops.

Fine mapping of a Phytophthora-resistance gene RpsWY in soybean (Glycine max L.) by high-throughput genome-wide sequencing.

Science.gov (United States)

Cheng, Yanbo; Ma, Qibin; Ren, Hailong; Xia, Qiuju; Song, Enliang; Tan, Zhiyuan; Li, Shuxian; Zhang, Gengyun; Nian, Hai

2017-05-01

Using a combination of phenotypic screening, genetic and statistical analyses, and high-throughput genome-wide sequencing, we have finely mapped a dominant Phytophthora resistance gene in soybean cultivar Wayao. Phytophthora root rot (PRR) caused by Phytophthora sojae is one of the most important soil-borne diseases in many soybean-production regions in the world. Identification of resistant gene(s) and incorporating them into elite varieties are an effective way for breeding to prevent soybean from being harmed by this disease. Two soybean populations of 191 F 2 individuals and 196 F 7:8 recombinant inbred lines (RILs) were developed to map Rps gene by crossing a susceptible cultivar Huachun 2 with the resistant cultivar Wayao. Genetic analysis of the F 2 population indicated that PRR resistance in Wayao was controlled by a single dominant gene, temporarily named RpsWY, which was mapped on chromosome 3. A high-density genetic linkage bin map was constructed using 3469 recombination bins of the RILs to explore the candidate genes by the high-throughput genome-wide sequencing. The results of genotypic analysis showed that the RpsWY gene was located in bin 401 between 4466230 and 4502773 bp on chromosome 3 through line 71 and 100 of the RILs. Four predicted genes (Glyma03g04350, Glyma03g04360, Glyma03g04370, and Glyma03g04380) were found at the narrowed region of 36.5 kb in bin 401. These results suggest that the high-throughput genome-wide resequencing is an effective method to fine map PRR candidate genes.

High-throughput sequencing of the T cell receptor β gene identifies aggressive early-stage mycosis fungoides.

Science.gov (United States)

de Masson, Adele; O'Malley, John T; Elco, Christopher P; Garcia, Sarah S; Divito, Sherrie J; Lowry, Elizabeth L; Tawa, Marianne; Fisher, David C; Devlin, Phillip M; Teague, Jessica E; Leboeuf, Nicole R; Kirsch, Ilan R; Robins, Harlan; Clark, Rachael A; Kupper, Thomas S

2018-05-09

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor β gene ( TCRB ) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

A high throughput DNA extraction method with high yield and quality

Directory of Open Access Journals (Sweden)

Xin Zhanguo

2012-07-01

Full Text Available Abstract Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome, and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L. Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

LncRNA Expression Profile of Human Thoracic Aortic Dissection by High-Throughput Sequencing.

Science.gov (United States)

Sun, Jie; Chen, Guojun; Jing, Yuanwen; He, Xiang; Dong, Jianting; Zheng, Junmeng; Zou, Meisheng; Li, Hairui; Wang, Shifei; Sun, Yili; Liao, Wangjun; Liao, Yulin; Feng, Li; Bin, Jianping

2018-01-01

In this study, the long non-coding RNA (lncRNA) expression profile in human thoracic aortic dissection (TAD), a highly lethal cardiovascular disease, was investigated. Human TAD (n=3) and normal aortic tissues (NA) (n=3) were examined by high-throughput sequencing. Bioinformatics analyses were performed to predict the roles of aberrantly expressed lncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to validate the results. A total of 269 lncRNAs (159 up-regulated and 110 down-regulated) and 2, 255 mRNAs (1 294 up-regulated and 961 down-regulated) were aberrantly expressed in human TAD (fold-change> 1.5, PTAD than in NA. The predicted binding motifs of three up-regulated lncRNAs (ENSG00000248508, ENSG00000226530, and EG00000259719) were correlated with up-regulated RUNX1 (R=0.982, PTAD. These findings suggest that lncRNAs are novel potential therapeutic targets for human TAD. © 2018 The Author(s). Published by S. Karger AG, Basel.

High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

Science.gov (United States)

Kretz, Colin A; Tomberg, Kärt; Van Esbroeck, Alexander; Yee, Andrew; Ginsburg, David

2018-02-12

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10 7 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

PipeCraft: Flexible open-source toolkit for bioinformatics analysis of custom high-throughput amplicon sequencing data.

Science.gov (United States)

Anslan, Sten; Bahram, Mohammad; Hiiesalu, Indrek; Tedersoo, Leho

2017-11-01

High-throughput sequencing methods have become a routine analysis tool in environmental sciences as well as in public and private sector. These methods provide vast amount of data, which need to be analysed in several steps. Although the bioinformatics may be applied using several public tools, many analytical pipelines allow too few options for the optimal analysis for more complicated or customized designs. Here, we introduce PipeCraft, a flexible and handy bioinformatics pipeline with a user-friendly graphical interface that links several public tools for analysing amplicon sequencing data. Users are able to customize the pipeline by selecting the most suitable tools and options to process raw sequences from Illumina, Pacific Biosciences, Ion Torrent and Roche 454 sequencing platforms. We described the design and options of PipeCraft and evaluated its performance by analysing the data sets from three different sequencing platforms. We demonstrated that PipeCraft is able to process large data sets within 24 hr. The graphical user interface and the automated links between various bioinformatics tools enable easy customization of the workflow. All analytical steps and options are recorded in log files and are easily traceable. © 2017 John Wiley & Sons Ltd.

Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing

KAUST Repository

Idris, Ali

2014-03-12

Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions. 2014 by the authors; licensee MDPI, Basel, Switzerland.

Viral Metagenomics: Analysis of Begomoviruses by Illumina High-Throughput Sequencing

Directory of Open Access Journals (Sweden)

Ali Idris

2014-03-01

Full Text Available Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes (genus, Begomovirus; family, Geminiviridae were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA. Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS. CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.

High Throughput Transcriptomics @ USEPA (Toxicology ...

Science.gov (United States)

The ideal chemical testing approach will provide complete coverage of all relevant toxicological responses. It should be sensitive and specific It should identify the mechanism/mode-of-action (with dose-dependence). It should identify responses relevant to the species of interest. Responses should ideally be translated into tissue-, organ-, and organism-level effects. It must be economical and scalable. Using a High Throughput Transcriptomics platform within US EPA provides broader coverage of biological activity space and toxicological MOAs and helps fill the toxicological data gap. Slide presentation at the 2016 ToxForum on using High Throughput Transcriptomics at US EPA for broader coverage biological activity space and toxicological MOAs.

High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

Science.gov (United States)

High throughput toxicokinetics (HTTK) is an approach that allows for rapid estimations of TK for hundreds of environmental chemicals. HTTK-based reverse dosimetry (i.e, reverse toxicokinetics or RTK) is used in order to convert high throughput in vitro toxicity screening (HTS) da...

CSReport: A New Computational Tool Designed for Automatic Analysis of Class Switch Recombination Junctions Sequenced by High-Throughput Sequencing.

Science.gov (United States)

Boyer, François; Boutouil, Hend; Dalloul, Iman; Dalloul, Zeinab; Cook-Moreau, Jeanne; Aldigier, Jean-Claude; Carrion, Claire; Herve, Bastien; Scaon, Erwan; Cogné, Michel; Péron, Sophie

2017-05-15

B cells ensure humoral immune responses due to the production of Ag-specific memory B cells and Ab-secreting plasma cells. In secondary lymphoid organs, Ag-driven B cell activation induces terminal maturation and Ig isotype class switch (class switch recombination [CSR]). CSR creates a virtually unique IgH locus in every B cell clone by intrachromosomal recombination between two switch (S) regions upstream of each C region gene. Amount and structural features of CSR junctions reveal valuable information about the CSR mechanism, and analysis of CSR junctions is useful in basic and clinical research studies of B cell functions. To provide an automated tool able to analyze large data sets of CSR junction sequences produced by high-throughput sequencing (HTS), we designed CSReport, a software program dedicated to support analysis of CSR recombination junctions sequenced with a HTS-based protocol (Ion Torrent technology). CSReport was assessed using simulated data sets of CSR junctions and then used for analysis of Sμ-Sα and Sμ-Sγ1 junctions from CH12F3 cells and primary murine B cells, respectively. CSReport identifies junction segment breakpoints on reference sequences and junction structure (blunt-ended junctions or junctions with insertions or microhomology). Besides the ability to analyze unprecedentedly large libraries of junction sequences, CSReport will provide a unified framework for CSR junction studies. Our results show that CSReport is an accurate tool for analysis of sequences from our HTS-based protocol for CSR junctions, thereby facilitating and accelerating their study. Copyright © 2017 by The American Association of Immunologists, Inc.

Temporal dynamics of soil microbial communities under different moisture regimes: high-throughput sequencing and bioinformatics analysis

Science.gov (United States)

Semenov, Mikhail; Zhuravleva, Anna; Semenov, Vyacheslav; Yevdokimov, Ilya; Larionova, Alla

2017-04-01

Recent climate scenarios predict not only continued global warming but also an increased frequency and intensity of extreme climatic events such as strong changes in temperature and precipitation regimes. Microorganisms are well known to be more sensitive to changes in environmental conditions than to other soil chemical and physical parameters. In this study, we determined the shifts in soil microbial community structure as well as indicative taxa in soils under three moisture regimes using high-throughput Illumina sequencing and range of bioinformatics approaches for the assessment of sequence data. Incubation experiments were performed in soil-filled (Greyic Phaeozems Albic) rhizoboxes with maize and without plants. Three contrasting moisture regimes were being simulated: 1) optimal wetting (OW), a watering 2-3 times per week to maintain soil moisture of 20-25% by weight; 2) periodic wetting (PW), with alternating periods of wetting and drought; and 3) constant insufficient wetting (IW), while soil moisture of 12% by weight was permanently maintained. Sampled fresh soils were homogenized, and the total DNA of three replicates was extracted using the FastDNA® SPIN kit for Soil. DNA replicates were combined in a pooled sample and the DNA was used for PCR with specific primers for the 16S V3 and V4 regions. In order to compare variability between different samples and replicates within a single sample, some DNA replicates treated separately. The products were purified and submitted to Illumina MiSeq sequencing. Sequence data were evaluated by alpha-diversity (Chao1 and Shannon H' diversity indexes), beta-diversity (UniFrac and Bray-Curtis dissimilarity), heatmap, tagcloud, and plot-bar analyses using the MiSeq Reporter Metagenomics Workflow and R packages (phyloseq, vegan, tagcloud). Shannon index varied in a rather narrow range (4.4-4.9) with the lowest values for microbial communities under PW treatment. Chao1 index varied from 385 to 480, being a more flexible

BioVLAB-MMIA-NGS: microRNA-mRNA integrated analysis using high-throughput sequencing data.

Science.gov (United States)

Chae, Heejoon; Rhee, Sungmin; Nephew, Kenneth P; Kim, Sun

2015-01-15

It is now well established that microRNAs (miRNAs) play a critical role in regulating gene expression in a sequence-specific manner, and genome-wide efforts are underway to predict known and novel miRNA targets. However, the integrated miRNA-mRNA analysis remains a major computational challenge, requiring powerful informatics systems and bioinformatics expertise. The objective of this study was to modify our widely recognized Web server for the integrated mRNA-miRNA analysis (MMIA) and its subsequent deployment on the Amazon cloud (BioVLAB-MMIA) to be compatible with high-throughput platforms, including next-generation sequencing (NGS) data (e.g. RNA-seq). We developed a new version called the BioVLAB-MMIA-NGS, deployed on both Amazon cloud and on a high-performance publicly available server called MAHA. By using NGS data and integrating various bioinformatics tools and databases, BioVLAB-MMIA-NGS offers several advantages. First, sequencing data is more accurate than array-based methods for determining miRNA expression levels. Second, potential novel miRNAs can be detected by using various computational methods for characterizing miRNAs. Third, because miRNA-mediated gene regulation is due to hybridization of an miRNA to its target mRNA, sequencing data can be used to identify many-to-many relationship between miRNAs and target genes with high accuracy. http://epigenomics.snu.ac.kr/biovlab_mmia_ngs/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome

Energy Technology Data Exchange (ETDEWEB)

Chhabra, S.R.; Butland, G.; Elias, D.; Chandonia, J.-M.; Fok, V.; Juba, T.; Gorur, A.; Allen, S.; Leung, C.-M.; Keller, K.; Reveco, S.; Zane, G.; Semkiw, E.; Prathapam, R.; Gold, B.; Singer, M.; Ouellet, M.; Sazakal, E.; Jorgens, D.; Price, M.; Witkowska, E.; Beller, H.; Hazen, T.C.; Biggin, M.; Auer, M.; Wall, J.; Keasling, J.

2011-07-15

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high- throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications including gene replacement and creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

Science.gov (United States)

Wan, Dongjin; Liu, Yongde; Niu, Zhenhua; Xiao, Shuhu; Li, Daorong

2016-02-01

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.

Highly multiplexed targeted DNA sequencing from single nuclei.

Science.gov (United States)

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

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, nois...... patterns of mutual exclusivity. These techniques, coupled with advances in high-throughput DNA sequencing, are enabling precision medicine approaches to the diagnosis and treatment of cancer....

A high-throughput screening approach to discovering good forms of biologically inspired visual representation.

Science.gov (United States)

Pinto, Nicolas; Doukhan, David; DiCarlo, James J; Cox, David D

2009-11-01

While many models of biological object recognition share a common set of "broad-stroke" properties, the performance of any one model depends strongly on the choice of parameters in a particular instantiation of that model--e.g., the number of units per layer, the size of pooling kernels, exponents in normalization operations, etc. Since the number of such parameters (explicit or implicit) is typically large and the computational cost of evaluating one particular parameter set is high, the space of possible model instantiations goes largely unexplored. Thus, when a model fails to approach the abilities of biological visual systems, we are left uncertain whether this failure is because we are missing a fundamental idea or because the correct "parts" have not been tuned correctly, assembled at sufficient scale, or provided with enough training. Here, we present a high-throughput approach to the exploration of such parameter sets, leveraging recent advances in stream processing hardware (high-end NVIDIA graphic cards and the PlayStation 3's IBM Cell Processor). In analogy to high-throughput screening approaches in molecular biology and genetics, we explored thousands of potential network architectures and parameter instantiations, screening those that show promising object recognition performance for further analysis. We show that this approach can yield significant, reproducible gains in performance across an array of basic object recognition tasks, consistently outperforming a variety of state-of-the-art purpose-built vision systems from the literature. As the scale of available computational power continues to expand, we argue that this approach has the potential to greatly accelerate progress in both artificial vision and our understanding of the computational underpinning of biological vision.

A high-throughput screening approach to discovering good forms of biologically inspired visual representation.

Directory of Open Access Journals (Sweden)

Nicolas Pinto

2009-11-01

Full Text Available While many models of biological object recognition share a common set of "broad-stroke" properties, the performance of any one model depends strongly on the choice of parameters in a particular instantiation of that model--e.g., the number of units per layer, the size of pooling kernels, exponents in normalization operations, etc. Since the number of such parameters (explicit or implicit is typically large and the computational cost of evaluating one particular parameter set is high, the space of possible model instantiations goes largely unexplored. Thus, when a model fails to approach the abilities of biological visual systems, we are left uncertain whether this failure is because we are missing a fundamental idea or because the correct "parts" have not been tuned correctly, assembled at sufficient scale, or provided with enough training. Here, we present a high-throughput approach to the exploration of such parameter sets, leveraging recent advances in stream processing hardware (high-end NVIDIA graphic cards and the PlayStation 3's IBM Cell Processor. In analogy to high-throughput screening approaches in molecular biology and genetics, we explored thousands of potential network architectures and parameter instantiations, screening those that show promising object recognition performance for further analysis. We show that this approach can yield significant, reproducible gains in performance across an array of basic object recognition tasks, consistently outperforming a variety of state-of-the-art purpose-built vision systems from the literature. As the scale of available computational power continues to expand, we argue that this approach has the potential to greatly accelerate progress in both artificial vision and our understanding of the computational underpinning of biological vision.

Identification of protoplast-isolation responsive microRNAs in Citrus reticulata Blanco by high-throughput sequencing.

Science.gov (United States)

Xu, Xiaoyong; Xu, Xiaoling; Zhou, Yipeng; Zeng, Shaohua; Kong, Weiwen

2017-01-01

Protoplast isolation is a stress-inducing process, during which a variety of physiological and molecular alterations take place. Such stress response affects the expression of totipotency of cultured protoplasts. MicroRNAs (miRNAs) play important roles in plant growth, development and stress responses. However, the underlying mechanism of miRNAs involved in the protoplast totipotency remains unclear. In this study, high-throughput sequencing technology was used to sequence two populations of small RNA from calli and callus-derived protoplasts in Citrus reticulata Blanco. A total of 67 known miRNAs from 35 families and 277 novel miRNAs were identified. Among these miRNAs, 18 known miRNAs and 64 novel miRNAs were identified by differentially expressed miRNAs (DEMs) analysis. The expression patterns of the eight DEMs were verified by qRT-PCR. Target prediction showed most targets of the miRNAs were transcription factors. The expression levels of half targets showed a negative correlation to those of the miRNAs. Furthermore, the physiological analysis showed high levels of antioxidant activities in isolated protoplasts. In short, our results indicated that miRNAs may play important roles in protoplast-isolation response.

Identification and characterization of microRNAs in Humulus lupulus using high-throughput sequencing and their response to Citrus bark cracking viroid (CBCVd) infection

Czech Academy of Sciences Publication Activity Database

Mishra, Ajay Kumar; Duraisamy, Ganesh Selvaraj; Matoušek, Jaroslav; Radišek, S.; Javornik, B.; Jakše, J.

2016-01-01

Roč. 17, č. 919 (2016) ISSN 1471-2164 R&D Projects: GA MŠk(CZ) LH14255 Institutional support: RVO:60077344 Keywords : Humulus lupulus * High-throughput sequencing * Citrus bark cracking viroid Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.729, year: 2016

Abundance and diversity of bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants revealed by high-throughput sequencing.

Directory of Open Access Journals (Sweden)

Zhu Wang

Full Text Available Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.

A ground-up approach to High Throughput Cloud Computing in High-Energy Physics

CERN Document Server

AUTHOR|(INSPIRE)INSPIRE-00245123; Ganis, Gerardo; Bagnasco, Stefano

The thesis explores various practical approaches in making existing High Throughput computing applications common in High Energy Physics work on cloud-provided resources, as well as opening the possibility for running new applications. The work is divided into two parts: firstly we describe the work done at the computing facility hosted by INFN Torino to entirely convert former Grid resources into cloud ones, eventually running Grid use cases on top along with many others in a more flexible way. Integration and conversion problems are duly described. The second part covers the development of solutions for automatizing the orchestration of cloud workers based on the load of a batch queue and the development of HEP applications based on ROOT's PROOF that can adapt at runtime to a changing number of workers.

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.

GlycoExtractor: a web-based interface for high throughput processing of HPLC-glycan data.

Science.gov (United States)

Artemenko, Natalia V; Campbell, Matthew P; Rudd, Pauline M

2010-04-05

Recently, an automated high-throughput HPLC platform has been developed that can be used to fully sequence and quantify low concentrations of N-linked sugars released from glycoproteins, supported by an experimental database (GlycoBase) and analytical tools (autoGU). However, commercial packages that support the operation of HPLC instruments and data storage lack platforms for the extraction of large volumes of data. The lack of resources and agreed formats in glycomics is now a major limiting factor that restricts the development of bioinformatic tools and automated workflows for high-throughput HPLC data analysis. GlycoExtractor is a web-based tool that interfaces with a commercial HPLC database/software solution to facilitate the extraction of large volumes of processed glycan profile data (peak number, peak areas, and glucose unit values). The tool allows the user to export a series of sample sets to a set of file formats (XML, JSON, and CSV) rather than a collection of disconnected files. This approach not only reduces the amount of manual refinement required to export data into a suitable format for data analysis but also opens the field to new approaches for high-throughput data interpretation and storage, including biomarker discovery and validation and monitoring of online bioprocessing conditions for next generation biotherapeutics.

Profile and Fate of Bacterial Pathogens in Sewage Treatment Plants Revealed by High-Throughput Metagenomic Approach.

Science.gov (United States)

Li, Bing; Ju, Feng; Cai, Lin; Zhang, Tong

2015-09-01

The broad-spectrum profile of bacterial pathogens and their fate in sewage treatment plants (STPs) were investigated using high-throughput sequencing based metagenomic approach. This novel approach could provide a united platform to standardize bacterial pathogen detection and realize direct comparison among different samples. Totally, 113 bacterial pathogen species were detected in eight samples including influent, effluent, activated sludge (AS), biofilm, and anaerobic digestion sludge with the abundances ranging from 0.000095% to 4.89%. Among these 113 bacterial pathogens, 79 species were reported in STPs for the first time. Specially, compared to AS in bulk mixed liquor, more pathogen species and higher total abundance were detected in upper foaming layer of AS. This suggests that the foaming layer of AS might impose more threat to onsite workers and citizens in the surrounding areas of STPs because pathogens in foaming layer are easily transferred into air and cause possible infections. The high removal efficiency (98.0%) of total bacterial pathogens suggests that AS treatment process is effective to remove most bacterial pathogens. Remarkable similarities of bacterial pathogen compositions between influent and human gut indicated that bacterial pathogen profiles in influents could well reflect the average bacterial pathogen communities of urban resident guts within the STP catchment area.

Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

Science.gov (United States)

Menzel, Ulrike; Greiff, Victor; Khan, Tarik A; Haessler, Ulrike; Hellmann, Ina; Friedensohn, Simon; Cook, Skylar C; Pogson, Mark; Reddy, Sai T

2014-01-01

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Science.gov (United States)

Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

2013-01-01

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet

Science.gov (United States)

State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet (Presented by Dr. Marilyn J. Aardema, Chief Scientific Advisor, Toxicology, Dr. Leon Stankowski, et. al. (6/28/2012)

Identification of microRNAs from Eugenia uniflora by high-throughput sequencing and bioinformatics analysis.

Science.gov (United States)

Guzman, Frank; Almerão, Mauricio P; Körbes, Ana P; Loss-Morais, Guilherme; Margis, Rogerio

2012-01-01

microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. Eugenia uniflora is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species. Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in Eugenia uniflora. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology. This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs.

A direct comparison of remote sensing approaches for high-throughput phenotyping in plant breeding

Directory of Open Access Journals (Sweden)

Maria Tattaris

2016-08-01

Full Text Available Remote sensing (RS of plant canopies permits non-intrusive, high-throughput monitoring of plant physiological characteristics. This study compared three RS approaches using a low flying UAV (unmanned aerial vehicle, with that of proximal sensing, and satellite-based imagery. Two physiological traits were considered, canopy temperature (CT and a vegetation index (NDVI, to determine the most viable approaches for large scale crop genetic improvement. The UAV-based platform achieves plot-level resolution while measuring several hundred plots in one mission via high-resolution thermal and multispectral imagery measured at altitudes of 30-100 m. The satellite measures multispectral imagery from an altitude of 770 km. Information was compared with proximal measurements using IR thermometers and an NDVI sensor at a distance of 0.5-1m above plots. For robust comparisons, CT and NDVI were assessed on panels of elite cultivars under irrigated and drought conditions, in different thermal regimes, and on un-adapted genetic resources under water deficit. Correlations between airborne data and yield/biomass at maturity were generally higher than equivalent proximal correlations. NDVI was derived from high-resolution satellite imagery for only larger sized plots (8.5 x 2.4 m due to restricted pixel density. Results support use of UAV-based RS techniques for high-throughput phenotyping for both precision and efficiency.

A high-throughput FTIR spectroscopy approach to assess adaptive variation in the chemical composition of pollen.

Science.gov (United States)

Zimmermann, Boris; Bağcıoğlu, Murat; Tafinstseva, Valeria; Kohler, Achim; Ohlson, Mikael; Fjellheim, Siri

2017-12-01

The two factors defining male reproductive success in plants are pollen quantity and quality, but our knowledge about the importance of pollen quality is limited due to methodological constraints. Pollen quality in terms of chemical composition may be either genetically fixed for high performance independent of environmental conditions, or it may be plastic to maximize reproductive output under different environmental conditions. In this study, we validated a new approach for studying the role of chemical composition of pollen in adaptation to local climate. The approach is based on high-throughput Fourier infrared (FTIR) characterization and biochemical interpretation of pollen chemical composition in response to environmental conditions. The study covered three grass species, Poa alpina , Anthoxanthum odoratum , and Festuca ovina . For each species, plants were grown from seeds of three populations with wide geographic and climate variation. Each individual plant was divided into four genetically identical clones which were grown in different controlled environments (high and low levels of temperature and nutrients). In total, 389 samples were measured using a high-throughput FTIR spectrometer. The biochemical fingerprints of pollen were species and population specific, and plastic in response to different environmental conditions. The response was most pronounced for temperature, influencing the levels of proteins, lipids, and carbohydrates in pollen of all species. Furthermore, there is considerable variation in plasticity of the chemical composition of pollen among species and populations. The use of high-throughput FTIR spectroscopy provides fast, cheap, and simple assessment of the chemical composition of pollen. In combination with controlled-condition growth experiments and multivariate analyses, FTIR spectroscopy opens up for studies of the adaptive role of pollen that until now has been difficult with available methodology. The approach can easily be

REDItools: high-throughput RNA editing detection made easy.

Science.gov (United States)

Picardi, Ernesto; Pesole, Graziano

2013-07-15

The reliable detection of RNA editing sites from massive sequencing data remains challenging and, although several methodologies have been proposed, no computational tools have been released to date. Here, we introduce REDItools a suite of python scripts to perform high-throughput investigation of RNA editing using next-generation sequencing data. REDItools are in python programming language and freely available at http://code.google.com/p/reditools/. ernesto.picardi@uniba.it or graziano.pesole@uniba.it Supplementary data are available at Bioinformatics online.

An improved high throughput sequencing method for studying oomycete communities

DEFF Research Database (Denmark)

Sapkota, Rumakanta; Nicolaisen, Mogens

2015-01-01

the usefulness of the method not only in soil DNA but also in a plant DNA background. In conclusion, we demonstrate a successful approach for pyrosequencing of oomycete communities using ITS1 as the barcode sequence with well-known primers for oomycete DNA amplification....... communities. Thewell-known primer sets ITS4, ITS6 and ITS7were used in the study in a semi-nested PCR approach to target the internal transcribed spacer (ITS) 1 of ribosomal DNA in a next generation sequencing protocol. These primers have been used in similar studies before, butwith limited success.......Wewere able to increase the proportion of retrieved oomycete sequences dramaticallymainly by increasing the annealing temperature during PCR. The optimized protocol was validated using three mock communities and the method was further evaluated using total DNA from 26 soil samples collected from different...

Optimization and high-throughput screening of antimicrobial peptides.

Science.gov (United States)

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data

Science.gov (United States)

Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data.

Science.gov (United States)

Miller, Mark P; Knaus, Brian J; Mullins, Thomas D; Haig, Susan M

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

High-throughput phenotyping and genomic selection: the frontiers of crop breeding converge.

Science.gov (United States)

Cabrera-Bosquet, Llorenç; Crossa, José; von Zitzewitz, Jarislav; Serret, María Dolors; Araus, José Luis

2012-05-01

Genomic selection (GS) and high-throughput phenotyping have recently been captivating the interest of the crop breeding community from both the public and private sectors world-wide. Both approaches promise to revolutionize the prediction of complex traits, including growth, yield and adaptation to stress. Whereas high-throughput phenotyping may help to improve understanding of crop physiology, most powerful techniques for high-throughput field phenotyping are empirical rather than analytical and comparable to genomic selection. Despite the fact that the two methodological approaches represent the extremes of what is understood as the breeding process (phenotype versus genome), they both consider the targeted traits (e.g. grain yield, growth, phenology, plant adaptation to stress) as a black box instead of dissecting them as a set of secondary traits (i.e. physiological) putatively related to the target trait. Both GS and high-throughput phenotyping have in common their empirical approach enabling breeders to use genome profile or phenotype without understanding the underlying biology. This short review discusses the main aspects of both approaches and focuses on the case of genomic selection of maize flowering traits and near-infrared spectroscopy (NIRS) and plant spectral reflectance as high-throughput field phenotyping methods for complex traits such as crop growth and yield. © 2012 Institute of Botany, Chinese Academy of Sciences.

High-throughput screening of small molecule libraries using SAMDI mass spectrometry.

Science.gov (United States)

Gurard-Levin, Zachary A; Scholle, Michael D; Eisenberg, Adam H; Mrksich, Milan

2011-07-11

High-throughput screening is a common strategy used to identify compounds that modulate biochemical activities, but many approaches depend on cumbersome fluorescent reporters or antibodies and often produce false-positive hits. The development of "label-free" assays addresses many of these limitations, but current approaches still lack the throughput needed for applications in drug discovery. This paper describes a high-throughput, label-free assay that combines self-assembled monolayers with mass spectrometry, in a technique called SAMDI, as a tool for screening libraries of 100,000 compounds in one day. This method is fast, has high discrimination, and is amenable to a broad range of chemical and biological applications.

Arioc: high-throughput read alignment with GPU-accelerated exploration of the seed-and-extend search space

Directory of Open Access Journals (Sweden)

Richard Wilton

2015-03-01

Full Text Available When computing alignments of DNA sequences to a large genome, a key element in achieving high processing throughput is to prioritize locations in the genome where high-scoring mappings might be expected. We formulated this task as a series of list-processing operations that can be efficiently performed on graphics processing unit (GPU hardware.We followed this approach in implementing a read aligner called Arioc that uses GPU-based parallel sort and reduction techniques to identify high-priority locations where potential alignments may be found. We then carried out a read-by-read comparison of Arioc’s reported alignments with the alignments found by several leading read aligners. With simulated reads, Arioc has comparable or better accuracy than the other read aligners we tested. With human sequencing reads, Arioc demonstrates significantly greater throughput than the other aligners we evaluated across a wide range of sensitivity settings. The Arioc software is available at https://github.com/RWilton/Arioc. It is released under a BSD open-source license.

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

Science.gov (United States)

Saeidi, Saman; McKain, Michael R; Kellogg, Elizabeth A

2018-03-08

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

High-throughput epitope identification for snakebite antivenom

DEFF Research Database (Denmark)

Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard

Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families.......Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individual...

Genetic profiles of cervical tumors by high-throughput sequencing for personalized medical care

International Nuclear Information System (INIS)

Muller, Etienne; Brault, Baptiste; Holmes, Allyson; Legros, Angelina; Jeannot, Emmanuelle; Campitelli, Maura; Rousselin, Antoine; Goardon, Nicolas; Frébourg, Thierry; Krieger, Sophie; Crouet, Hubert; Nicolas, Alain; Sastre, Xavier; Vaur, Dominique; Castéra, Laurent

2015-01-01

Cancer treatment is facing major evolution since the advent of targeted therapies. Building genetic profiles could predict sensitivity or resistance to these therapies and highlight disease-specific abnormalities, supporting personalized patient care. In the context of biomedical research and clinical diagnosis, our laboratory has developed an oncogenic panel comprised of 226 genes and a dedicated bioinformatic pipeline to explore somatic mutations in cervical carcinomas, using high-throughput sequencing. Twenty-nine tumors were sequenced for exons within 226 genes. The automated pipeline used includes a database and a filtration system dedicated to identifying mutations of interest and excluding false positive and germline mutations. One-hundred and seventy-six total mutational events were found among the 29 tumors. Our cervical tumor mutational landscape shows that most mutations are found in PIK3CA (E545K, E542K) and KRAS (G12D, G13D) and others in FBXW7 (R465C, R505G, R479Q). Mutations have also been found in ALK (V1149L, A1266T) and EGFR (T259M). These results showed that 48% of patients display at least one deleterious mutation in genes that have been already targeted by the Food and Drug Administration approved therapies. Considering deleterious mutations, 59% of patients could be eligible for clinical trials. Sequencing hundreds of genes in a clinical context has become feasible, in terms of time and cost. In the near future, such an analysis could be a part of a battery of examinations along the diagnosis and treatment of cancer, helping to detect sensitivity or resistance to targeted therapies and allow advancements towards personalized oncology

High Throughput T Epitope Mapping and Vaccine Development

Directory of Open Access Journals (Sweden)

Giuseppina Li Pira

2010-01-01

Full Text Available Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th and by cytolytic T lymphocytes (CTL is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.

High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yonghua [Iowa State Univ., Ames, IA (United States)

2000-01-01

Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences.

Science.gov (United States)

Alcantara, Luiz Carlos Junior; Cassol, Sharon; Libin, Pieter; Deforche, Koen; Pybus, Oliver G; Van Ranst, Marc; Galvão-Castro, Bernardo; Vandamme, Anne-Mieke; de Oliveira, Tulio

2009-07-01

Human immunodeficiency virus type-1 (HIV-1), hepatitis B and C and other rapidly evolving viruses are characterized by extremely high levels of genetic diversity. To facilitate diagnosis and the development of prevention and treatment strategies that efficiently target the diversity of these viruses, and other pathogens such as human T-lymphotropic virus type-1 (HTLV-1), human herpes virus type-8 (HHV8) and human papillomavirus (HPV), we developed a rapid high-throughput-genotyping system. The method involves the alignment of a query sequence with a carefully selected set of pre-defined reference strains, followed by phylogenetic analysis of multiple overlapping segments of the alignment using a sliding window. Each segment of the query sequence is assigned the genotype and sub-genotype of the reference strain with the highest bootstrap (>70%) and bootscanning (>90%) scores. Results from all windows are combined and displayed graphically using color-coded genotypes. The new Virus-Genotyping Tools provide accurate classification of recombinant and non-recombinant viruses and are currently being assessed for their diagnostic utility. They have incorporated into several HIV drug resistance algorithms including the Stanford (http://hivdb.stanford.edu) and two European databases (http://www.umcutrecht.nl/subsite/spread-programme/ and http://www.hivrdb.org.uk/) and have been successfully used to genotype a large number of sequences in these and other databases. The tools are a PHP/JAVA web application and are freely accessible on a number of servers including: http://bioafrica.mrc.ac.za/rega-genotype/html/, http://lasp.cpqgm.fiocruz.br/virus-genotype/html/, http://jose.med.kuleuven.be/genotypetool/html/.

Bioassessment of a Drinking Water Reservoir Using Plankton: High Throughput Sequencing vs. Traditional Morphological Method

Directory of Open Access Journals (Sweden)

Wanli Gao

2018-01-01

Full Text Available Drinking water safety is increasingly perceived as one of the top global environmental issues. Plankton has been commonly used as a bioindicator for water quality in lakes and reservoirs. Recently, DNA sequencing technology has been applied to bioassessment. In this study, we compared the effectiveness of the 16S and 18S rRNA high throughput sequencing method (HTS and the traditional optical microscopy method (TOM in the bioassessment of drinking water quality. Five stations reflecting different habitats and hydrological conditions in Danjiangkou Reservoir, one of the largest drinking water reservoirs in Asia, were sampled May 2016. Non-metric multi-dimensional scaling (NMDS analysis showed that plankton assemblages varied among the stations and the spatial patterns revealed by the two methods were consistent. The correlation between TOM and HTS in a symmetric Procrustes analysis was 0.61, revealing overall good concordance between the two methods. Procrustes analysis also showed that site-specific differences between the two methods varied among the stations. Station Heijizui (H, a site heavily influenced by two tributaries, had the largest difference while station Qushou (Q, a confluence site close to the outlet dam, had the smallest difference between the two methods. Our results show that DNA sequencing has the potential to provide consistent identification of taxa, and reliable bioassessment in a long-term biomonitoring and assessment program for drinking water reservoirs.

Evaluation of the microbial diversity in amyotrophic lateral sclerosis using high-throughput sequencing

Directory of Open Access Journals (Sweden)

Xin Fang

2016-09-01

Full Text Available More and more evidences indicate that diseases of the central nervous system (CNS have been seriously affected by faecal microbes. However, little work is done to explore interaction between amyotrophic lateral sclerosis (ALS and faecal microbes. In the present study, high-throughput sequencing method was used to compare the intestinal microbial diversity of healthy people and ALS patients. The principal coordinate analysis (PCoA, Venn and unweighted pair-group method using arithmetic averages (UPGMA showed an obvious microbial changes between healthy people (group H and ALS patients (group A, and the average ratios of Bacteroides, Faecalibacterium, Anaerostipes, Prevotella, Escherichia and Lachnospira at genus level between ALS patients and healthy people were 0.78, 2.18, 3.41, 0.35, 0.79 and 13.07. Furthermore, the decreased Firmicutes/Bacteroidetes ratio at phylum level using LEfSE (LDA >4.0, together with the significant increased genus Dorea (harmful microorganisms and significant reduced genus Oscillibacter, Anaerostipes, Lachnospiraceae (beneficial microorganisms in ALS patients, indicated that the imbalance in intestinal microflora constitution had a strong association with the pathogenesis of ALS.

Evaluation of the Microbial Diversity in Amyotrophic Lateral Sclerosis Using High-Throughput Sequencing.

Science.gov (United States)

Fang, Xin; Wang, Xin; Yang, Shaoguo; Meng, Fanjing; Wang, Xiaolei; Wei, Hua; Chen, Tingtao

2016-01-01

More and more evidences indicate that diseases of the central nervous system have been seriously affected by fecal microbes. However, little work is done to explore interaction between amyotrophic lateral sclerosis (ALS) and fecal microbes. In the present study, high-throughput sequencing method was used to compare the intestinal microbial diversity of healthy people and ALS patients. The principal coordinate analysis, Venn and unweighted pair-group method using arithmetic averages (UPGMA) showed an obvious microbial changes between healthy people (group H) and ALS patients (group A), and the average ratios of Bacteroides , Faecalibacterium , Anaerostipes , Prevotella , Escherichia , and Lachnospira at genus level between ALS patients and healthy people were 0.78, 2.18, 3.41, 0.35, 0.79, and 13.07. Furthermore, the decreased Firmicutes/Bacteroidetes ratio at phylum level using LEfSE (LDA > 4.0), together with the significant increased genus Dorea (harmful microorganisms) and significant reduced genus Oscillibacter , Anaerostipes , Lachnospiraceae (beneficial microorganisms) in ALS patients, indicated that the imbalance in intestinal microflora constitution had a strong association with the pathogenesis of ALS.

The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.

Directory of Open Access Journals (Sweden)

John K Davies

Full Text Available Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1 as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11 are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

High-throughput open source computational methods for genetics and genomics

NARCIS (Netherlands)

Prins, J.C.P.

2015-01-01

Biology is increasingly data driven by virtue of the development of high-throughput technologies, such as DNA and RNA sequencing. Computational biology and bioinformatics are scientific disciplines that cross-over between the disciplines of biology, informatics and statistics; which is clearly

Who's for dinner? High-throughput sequencing reveals bat dietary differentiation in a biodiversity hotspot where prey taxonomy is largely undescribed.

Science.gov (United States)

Burgar, Joanna M; Murray, Daithi C; Craig, Michael D; Haile, James; Houston, Jayne; Stokes, Vicki; Bunce, Michael

2014-08-01

Effective management and conservation of biodiversity requires understanding of predator-prey relationships to ensure the continued existence of both predator and prey populations. Gathering dietary data from predatory species, such as insectivorous bats, often presents logistical challenges, further exacerbated in biodiversity hot spots because prey items are highly speciose, yet their taxonomy is largely undescribed. We used high-throughput sequencing (HTS) and bioinformatic analyses to phylogenetically group DNA sequences into molecular operational taxonomic units (MOTUs) to examine predator-prey dynamics of three sympatric insectivorous bat species in the biodiversity hotspot of south-western Australia. We could only assign between 4% and 20% of MOTUs to known genera or species, depending on the method used, underscoring the importance of examining dietary diversity irrespective of taxonomic knowledge in areas lacking a comprehensive genetic reference database. MOTU analysis confirmed that resource partitioning occurred, with dietary divergence positively related to the ecomorphological divergence of the three bat species. We predicted that bat species' diets would converge during times of high energetic requirements, that is, the maternity season for females and the mating season for males. There was an interactive effect of season on female, but not male, bat species' diets, although small sample sizes may have limited our findings. Contrary to our predictions, females of two ecomorphologically similar species showed dietary convergence during the mating season rather than the maternity season. HTS-based approaches can help elucidate complex predator-prey relationships in highly speciose regions, which should facilitate the conservation of biodiversity in genetically uncharacterized areas, such as biodiversity hotspots. © 2013 John Wiley & Sons Ltd.

Insight into the transcriptome of Arthrobotrys conoides using high throughput sequencing.

Science.gov (United States)

Ramesh, Pandit; Reena, Patel; Amitbikram, Mohapatra; Chaitanya, Joshi; Anju, Kunjadia

2015-12-01

Arthrobotrys conoides is a nematode-trapping fungus belonging to Orbiliales, Ascomycota group, and traps prey nematodes by means of adhesive network. Fungus has a potential to be used as a biocontrol agent against plant parasitic nematodes. In the present study, we characterized the transcriptome of A. conoides using high-throughput sequencing technology and characterized its virulence unigenes. Total 7,255 cDNA contigs with an average length of 425 bp were generated and 6184 (61.81%) transcripts were functionally annotated and characterized. Majority of unigenes were found analogous to the genes of plant pathogenic fungi. A total of 1749 transcripts were found to be orthologous with eukaryotic proteins of KOG database. Several carbohydrate active enzymes and peptidases were identified. We also analyzed classically and nonclassically secreted proteins and confirmed by BLASTP against fungal secretome database. A total of 916 contigs were analogous to 556 unique proteins of Pathogen Host Interaction (PHI) database. Further, we identified 91 unigenes homologous to the database of fungal virulence factor (DFVF). A total of 104 putative protein kinases coding transcripts were identified by BLASTP against KinBase database, which are major players in signaling pathways. This study provides a comprehensive look at the transcriptome of A. conoides and the identified unigenes might have a role in catching and killing prey nematodes by A. conoides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

Science.gov (United States)

Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

ImmuneDB: a system for the analysis and exploration of high-throughput adaptive immune receptor sequencing data.

Science.gov (United States)

Rosenfeld, Aaron M; Meng, Wenzhao; Luning Prak, Eline T; Hershberg, Uri

2017-01-15

As high-throughput sequencing of B cells becomes more common, the need for tools to analyze the large quantity of data also increases. This article introduces ImmuneDB, a system for analyzing vast amounts of heavy chain variable region sequences and exploring the resulting data. It can take as input raw FASTA/FASTQ data, identify genes, determine clones, construct lineages, as well as provide information such as selection pressure and mutation analysis. It uses an industry leading database, MySQL, to provide fast analysis and avoid the complexities of using error prone flat-files. ImmuneDB is freely available at http://immunedb.comA demo of the ImmuneDB web interface is available at: http://immunedb.com/demo CONTACT: Uh25@drexel.eduSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Discovery of precursor and mature microRNAs and their putative gene targets using high-throughput sequencing in pineapple (Ananas comosus var. comosus).

Science.gov (United States)

Yusuf, Noor Hydayaty Md; Ong, Wen Dee; Redwan, Raimi Mohamed; Latip, Mariam Abd; Kumar, S Vijay

2015-10-15

MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that negatively regulate gene expression, resulting in the silencing of target mRNA transcripts through mRNA cleavage or translational inhibition. MiRNAs play significant roles in various biological and physiological processes in plants. However, the miRNA-mediated gene regulatory network in pineapple, the model tropical non-climacteric fruit, remains largely unexplored. Here, we report a complete list of pineapple mature miRNAs obtained from high-throughput small RNA sequencing and precursor miRNAs (pre-miRNAs) obtained from ESTs. Two small RNA libraries were constructed from pineapple fruits and leaves, respectively, using Illumina's Solexa technology. Sequence similarity analysis using miRBase revealed 579,179 reads homologous to 153 miRNAs from 41 miRNA families. In addition, a pineapple fruit transcriptome library consisting of approximately 30,000 EST contigs constructed using Solexa sequencing was used for the discovery of pre-miRNAs. In all, four pre-miRNAs were identified (MIR156, MIR399, MIR444 and MIR2673). Furthermore, the same pineapple transcriptome was used to dissect the function of the miRNAs in pineapple by predicting their putative targets in conjunction with their regulatory networks. In total, 23 metabolic pathways were found to be regulated by miRNAs in pineapple. The use of high-throughput sequencing in pineapples to unveil the presence of miRNAs and their regulatory pathways provides insight into the repertoire of miRNA regulation used exclusively in this non-climacteric model plant. Copyright © 2015 Elsevier B.V. All rights reserved.

High-Throughput Tabular Data Processor - Platform independent graphical tool for processing large data sets.

Science.gov (United States)

Madanecki, Piotr; Bałut, Magdalena; Buckley, Patrick G; Ochocka, J Renata; Bartoszewski, Rafał; Crossman, David K; Messiaen, Ludwine M; Piotrowski, Arkadiusz

2018-01-01

High-throughput technologies generate considerable amount of data which often requires bioinformatic expertise to analyze. Here we present High-Throughput Tabular Data Processor (HTDP), a platform independent Java program. HTDP works on any character-delimited column data (e.g. BED, GFF, GTF, PSL, WIG, VCF) from multiple text files and supports merging, filtering and converting of data that is produced in the course of high-throughput experiments. HTDP can also utilize itemized sets of conditions from external files for complex or repetitive filtering/merging tasks. The program is intended to aid global, real-time processing of large data sets using a graphical user interface (GUI). Therefore, no prior expertise in programming, regular expression, or command line usage is required of the user. Additionally, no a priori assumptions are imposed on the internal file composition. We demonstrate the flexibility and potential of HTDP in real-life research tasks including microarray and massively parallel sequencing, i.e. identification of disease predisposing variants in the next generation sequencing data as well as comprehensive concurrent analysis of microarray and sequencing results. We also show the utility of HTDP in technical tasks including data merge, reduction and filtering with external criteria files. HTDP was developed to address functionality that is missing or rudimentary in other GUI software for processing character-delimited column data from high-throughput technologies. Flexibility, in terms of input file handling, provides long term potential functionality in high-throughput analysis pipelines, as the program is not limited by the currently existing applications and data formats. HTDP is available as the Open Source software (https://github.com/pmadanecki/htdp).

Diversity and Structure of Diazotrophic Communities in Mangrove Rhizosphere, Revealed by High-Throughput Sequencing.

Science.gov (United States)

Zhang, Yanying; Yang, Qingsong; Ling, Juan; Van Nostrand, Joy D; Shi, Zhou; Zhou, Jizhong; Dong, Junde

2017-01-01

Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata , Avicennia marina , and Ceriops tagal , was undertaken using high - throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. The Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.

Diversity and Structure of Diazotrophic Communities in Mangrove Rhizosphere, Revealed by High-Throughput Sequencing

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Yanying Zhang

2017-10-01

Full Text Available Diazotrophic communities make an essential contribution to the productivity through providing new nitrogen. However, knowledge of the roles that both mangrove tree species and geochemical parameters play in shaping mangove rhizosphere diazotrophic communities is still elusive. Here, a comprehensive examination of the diversity and structure of microbial communities in the rhizospheres of three mangrove species, Rhizophora apiculata, Avicennia marina, and Ceriops tagal, was undertaken using high-throughput sequencing of the 16S rRNA and nifH genes. Our results revealed a great diversity of both the total microbial composition and the diazotrophic composition specifically in the mangrove rhizosphere. Deltaproteobacteria and Gammaproteobacteria were both ubiquitous and dominant, comprising an average of 45.87 and 86.66% of total microbial and diazotrophic communities, respectively. Sulfate-reducing bacteria belonging to the Desulfobacteraceae and Desulfovibrionaceae were the dominant diazotrophs. Community statistical analyses suggested that both mangrove tree species and additional environmental variables played important roles in shaping total microbial and potential diazotroph communities in mangrove rhizospheres. In contrast to the total microbial community investigated by analysis of 16S rRNA gene sequences, most of the dominant diazotrophic groups identified by nifH gene sequences were significantly different among mangrove species. The dominant diazotrophs of the family Desulfobacteraceae were positively correlated with total phosphorus, but negatively correlated with the nitrogen to phosphorus ratio. The Pseudomonadaceae were positively correlated with the concentration of available potassium, suggesting that diazotrophs potentially play an important role in biogeochemical cycles, such as those of nitrogen, phosphorus, sulfur, and potassium, in the mangrove ecosystem.

Comparative analysis of transcriptomes in aerial stems and roots of Ephedra sinica based on high-throughput mRNA sequencing

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Taketo Okada

2016-12-01

Full Text Available Ephedra plants are taxonomically classified as gymnosperms, and are medicinally important as the botanical origin of crude drugs and as bioresources that contain pharmacologically active chemicals. Here we show a comparative analysis of the transcriptomes of aerial stems and roots of Ephedra sinica based on high-throughput mRNA sequencing by RNA-Seq. De novo assembly of short cDNA sequence reads generated 23,358, 13,373, and 28,579 contigs longer than 200 bases from aerial stems, roots, or both aerial stems and roots, respectively. The presumed functions encoded by these contig sequences were annotated by BLAST (blastx. Subsequently, these contigs were classified based on gene ontology slims, Enzyme Commission numbers, and the InterPro database. Furthermore, comparative gene expression analysis was performed between aerial stems and roots. These transcriptome analyses revealed differences and similarities between the transcriptomes of aerial stems and roots in E. sinica. Deep transcriptome sequencing of Ephedra should open the door to molecular biological studies based on the entire transcriptome, tissue- or organ-specific transcriptomes, or targeted genes of interest.

Molecular Approaches for High Throughput Detection and Quantification of Genetically Modified Crops: A Review

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Ibrahim B. Salisu

2017-10-01

Full Text Available As long as the genetically modified crops are gaining attention globally, their proper approval and commercialization need accurate and reliable diagnostic methods for the transgenic content. These diagnostic techniques are mainly divided into two major groups, i.e., identification of transgenic (1 DNA and (2 proteins from GMOs and their products. Conventional methods such as PCR (polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA were routinely employed for DNA and protein based quantification respectively. Although, these Techniques (PCR and ELISA are considered as significantly convenient and productive, but there is need for more advance technologies that allow for high throughput detection and the quantification of GM event as the production of more complex GMO is increasing day by day. Therefore, recent approaches like microarray, capillary gel electrophoresis, digital PCR and next generation sequencing are more promising due to their accuracy and precise detection of transgenic contents. The present article is a brief comparative study of all such detection techniques on the basis of their advent, feasibility, accuracy, and cost effectiveness. However, these emerging technologies have a lot to do with detection of a specific event, contamination of different events and determination of fusion as well as stacked gene protein are the critical issues to be addressed in future.

High Resolution Melting (HRM for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

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Marcin Słomka

2017-11-01

Full Text Available High resolution melting (HRM is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs. This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

High Resolution Melting (HRM) for High-Throughput Genotyping—Limitations and Caveats in Practical Case Studies

Science.gov (United States)

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz

2017-01-01

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup. PMID:29099791

High Resolution Melting (HRM) for High-Throughput Genotyping-Limitations and Caveats in Practical Case Studies.

Science.gov (United States)

Słomka, Marcin; Sobalska-Kwapis, Marta; Wachulec, Monika; Bartosz, Grzegorz; Strapagiel, Dominik

2017-11-03

High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

High-throughput verification of transcriptional starting sites by Deep-RACE

DEFF Research Database (Denmark)

Olivarius, Signe; Plessy, Charles; Carninci, Piero

2009-01-01

We present a high-throughput method for investigating the transcriptional starting sites of genes of interest, which we named Deep-RACE (Deep–rapid amplification of cDNA ends). Taking advantage of the latest sequencing technology, it allows the parallel analysis of multiple genes and is free...

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

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Chao Shiaoman

2011-01-01

Full Text Available Abstract Background Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat. Results Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM analysis. Of these, 52 (54% were polymorphic between parents of the Ogle1040 × TAM O-301 (OT mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry. Conclusions The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide

High throughput generation and trapping of individual agarose microgel using microfluidic approach

KAUST Repository

Shi, Yang

2013-02-28

Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering. © 2013 Springer-Verlag Berlin Heidelberg.

High throughput generation and trapping of individual agarose microgel using microfluidic approach

KAUST Repository

Shi, Yang; Gao, Xinghua; Chen, Longqing; Zhang, Min; Ma, Jingyun; Zhang, Xixiang; Qin, Jianhua

2013-01-01

Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering. © 2013 Springer-Verlag Berlin Heidelberg.

A novel library-independent approach based on high-throughput cultivation in Bioscreen and fingerprinting by FTIR spectroscopy for microbial source tracking in food industry.

Science.gov (United States)

Shapaval, V; Møretrø, T; Wold Åsli, A; Suso, H P; Schmitt, J; Lillehaug, D; Kohler, A

2017-05-01

Microbiological source tracking (MST) for food industry is a rapid growing area of research and technology development. In this paper, a new library-independent approach for MST is presented. It is based on a high-throughput liquid microcultivation and FTIR spectroscopy. In this approach, FTIR spectra obtained from micro-organisms isolated along the production line and a product are compared to each other. We tested and evaluated the new source tracking approach by simulating a source tracking situation. In this simulation study, a selection of 20 spoilage mould strains from a total of six genera (Alternaria, Aspergillus, Mucor, Paecilomyces, Peyronellaea and Phoma) was used. The simulation of the source tracking situation showed that 80-100% of the sources could be correctly identified with respect to genus/species level. When performing source tracking simulations, the FTIR identification diverged for Phoma glomerata strain in the reference collection. When reidentifying the strain by sequencing, it turned out that the strain was a Peyronellaea arachidicola. The obtained results demonstrated that the proposed approach is a versatile tool for identifying sources of microbial contamination. Thus, it has a high potential for routine control in the food industry due to low costs and analysis time. The source tracking of fungal contamination in the food industry is an important aspect of food safety. Currently, all available methods are time consuming and require the use of a reference library that may limit the accuracy of the identification. In this study, we report for the first time, a library-independent FTIR spectroscopic approach for MST of fungal contamination along the food production line. It combines high-throughput microcultivation and FTIR spectroscopy and is specific on the genus and species level. Therefore, such an approach possesses great importance for food safety control in food industry. © 2016 The Society for Applied Microbiology.

A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma

Science.gov (United States)

Xia, Hui; Strachan, Briony C.; Gifford, Sean C.; Shevkoplyas, Sergey S.

2016-10-01

Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current ‘leukoreduction’ filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional ‘dead end’ filtration. To address these limitations, here we demonstrate our newly-developed ‘controlled incremental filtration’ (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.

Fluorescence-based high-throughput screening of dicer cleavage activity.

Science.gov (United States)

Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

2014-03-01

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

Preliminary High-Throughput Metagenome Assembly

Energy Technology Data Exchange (ETDEWEB)

Dusheyko, Serge; Furman, Craig; Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

2007-03-26

Metagenome data sets present a qualitatively different assembly problem than traditional single-organism whole-genome shotgun (WGS) assembly. The unique aspects of such projects include the presence of a potentially large number of distinct organisms and their representation in the data set at widely different fractions. In addition, multiple closely related strains could be present, which would be difficult to assemble separately. Failure to take these issues into account can result in poor assemblies that either jumble together different strains or which fail to yield useful results. The DOE Joint Genome Institute has sequenced a number of metagenomic projects and plans to considerably increase this number in the coming year. As a result, the JGI has a need for high-throughput tools and techniques for handling metagenome projects. We present the techniques developed to handle metagenome assemblies in a high-throughput environment. This includes a streamlined assembly wrapper, based on the JGI?s in-house WGS assembler, Jazz. It also includes the selection of sensible defaults targeted for metagenome data sets, as well as quality control automation for cleaning up the raw results. While analysis is ongoing, we will discuss preliminary assessments of the quality of the assembly results (http://fames.jgi-psf.org).

High-throughput determination of RNA structure by proximity ligation.

Science.gov (United States)

Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

2015-09-01

We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

Human papillomavirus detection using the Abbott RealTime high-risk HPV tests compared with conventional nested PCR coupled to high-throughput sequencing of amplification products in cervical smear specimens from a Gabonese female population.

Science.gov (United States)

Moussavou-Boundzanga, Pamela; Koumakpayi, Ismaël Hervé; Labouba, Ingrid; Leroy, Eric M; Belembaogo, Ernest; Berthet, Nicolas

2017-12-21

Cervical cancer is the fourth most common malignancy in women worldwide. However, screening with human papillomavirus (HPV) molecular tests holds promise for reducing cervical cancer incidence and mortality in low- and middle-income countries. The performance of the Abbott RealTime High-Risk HPV test (AbRT) was evaluated in 83 cervical smear specimens and compared with a conventional nested PCR coupled to high-throughput sequencing (HTS) to identify the amplicons. The AbRT assay detected at least one HPV genotype in 44.57% of women regardless of the grade of cervical abnormalities. Except for one case, good concordance was observed for the genotypes detected with the AbRT assay in the high-risk HPV category determined with HTS of the amplicon generated by conventional nested PCR. The AbRT test is an easy and reliable molecular tool and was as sensitive as conventional nested PCR in cervical smear specimens for detection HPVs associated with high-grade lesions. Moreover, sequencing amplicons using an HTS approach effectively identified the genotype of the hrHPV identified with the AbRT test.

Low Complexity Approach for High Throughput Belief-Propagation based Decoding of LDPC Codes

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BOT, A.

2013-11-01

Full Text Available The paper proposes a low complexity belief propagation (BP based decoding algorithm for LDPC codes. In spite of the iterative nature of the decoding process, the proposed algorithm provides both reduced complexity and increased BER performances as compared with the classic min-sum (MS algorithm, generally used for hardware implementations. Linear approximations of check-nodes update function are used in order to reduce the complexity of the BP algorithm. Considering this decoding approach, an FPGA based hardware architecture is proposed for implementing the decoding algorithm, aiming to increase the decoder throughput. FPGA technology was chosen for the LDPC decoder implementation, due to its parallel computation and reconfiguration capabilities. The obtained results show improvements regarding decoding throughput and BER performances compared with state-of-the-art approaches.

High-throughput sequencing of plasma microRNA in chronic fatigue syndrome/myalgic encephalomyelitis.

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Ekua W Brenu

Full Text Available BACKGROUND: MicroRNAs (miRNAs are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME. The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. RESULTS: Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p in the CFS/ME patients. CONCLUSION: Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers.

High-Throughput Sequencing of Plasma MicroRNA in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Science.gov (United States)

Brenu, Ekua W.; Ashton, Kevin J.; Batovska, Jana; Staines, Donald R.; Marshall-Gradisnik, Sonya M.

2014-01-01

Background MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. Results Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p) in the CFS/ME patients. Conclusion Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers. PMID:25238588

An integrated tool to study MHC region: accurate SNV detection and HLA genes typing in human MHC region using targeted high-throughput sequencing.

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Hongzhi Cao

Full Text Available The major histocompatibility complex (MHC is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that ∼97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community.

A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries.

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David Skurnik

Full Text Available High-throughput sequencing of transposon (Tn libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian

Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.

Science.gov (United States)

Marine, Rachel; Polson, Shawn W; Ravel, Jacques; Hatfull, Graham; Russell, Daniel; Sullivan, Matthew; Syed, Fraz; Dumas, Michael; Wommack, K Eric

2011-11-01

Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.

MicroRNA from Moringa oleifera: Identification by High Throughput Sequencing and Their Potential Contribution to Plant Medicinal Value.

Science.gov (United States)

Pirrò, Stefano; Zanella, Letizia; Kenzo, Maurice; Montesano, Carla; Minutolo, Antonella; Potestà, Marina; Sobze, Martin Sanou; Canini, Antonella; Cirilli, Marco; Muleo, Rosario; Colizzi, Vittorio; Galgani, Andrea

2016-01-01

Moringa oleifera is a widespread plant with substantial nutritional and medicinal value. We postulated that microRNAs (miRNAs), which are endogenous, noncoding small RNAs regulating gene expression at the post-transcriptional level, might contribute to the medicinal properties of plants of this species after ingestion into human body, regulating human gene expression. However, the knowledge is scarce about miRNA in Moringa. Furthermore, in order to test the hypothesis on the pharmacological potential properties of miRNA, we conducted a high-throughput sequencing analysis using the Illumina platform. A total of 31,290,964 raw reads were produced from a library of small RNA isolated from M. oleifera seeds. We identified 94 conserved and two novel miRNAs that were validated by qRT-PCR assays. Results from qRT-PCR trials conducted on the expression of 20 Moringa miRNA showed that are conserved across multiple plant species as determined by their detection in tissue of other common crop plants. In silico analyses predicted target genes for the conserved miRNA that in turn allowed to relate the miRNAs to the regulation of physiological processes. Some of the predicted plant miRNAs have functional homology to their mammalian counterparts and regulated human genes when they were transfected into cell lines. To our knowledge, this is the first report of discovering M. oleifera miRNAs based on high-throughput sequencing and bioinformatics analysis and we provided new insight into a potential cross-species control of human gene expression. The widespread cultivation and consumption of M. oleifera, for nutritional and medicinal purposes, brings humans into close contact with products and extracts of this plant species. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for beneficial properties of this valuable species.

Two-stage clustering (TSC: a pipeline for selecting operational taxonomic units for the high-throughput sequencing of PCR amplicons.

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Xiao-Tao Jiang

Full Text Available Clustering 16S/18S rRNA amplicon sequences into operational taxonomic units (OTUs is a critical step for the bioinformatic analysis of microbial diversity. Here, we report a pipeline for selecting OTUs with a relatively low computational demand and a high degree of accuracy. This pipeline is referred to as two-stage clustering (TSC because it divides tags into two groups according to their abundance and clusters them sequentially. The more abundant group is clustered using a hierarchical algorithm similar to that in ESPRIT, which has a high degree of accuracy but is computationally costly for large datasets. The rarer group, which includes the majority of tags, is then heuristically clustered to improve efficiency. To further improve the computational efficiency and accuracy, two preclustering steps are implemented. To maintain clustering accuracy, all tags are grouped into an OTU depending on their pairwise Needleman-Wunsch distance. This method not only improved the computational efficiency but also mitigated the spurious OTU estimation from 'noise' sequences. In addition, OTUs clustered using TSC showed comparable or improved performance in beta-diversity comparisons compared to existing OTU selection methods. This study suggests that the distribution of sequencing datasets is a useful property for improving the computational efficiency and increasing the clustering accuracy of the high-throughput sequencing of PCR amplicons. The software and user guide are freely available at http://hwzhoulab.smu.edu.cn/paperdata/.

High-throughput GPU-based LDPC decoding

Science.gov (United States)

Chang, Yang-Lang; Chang, Cheng-Chun; Huang, Min-Yu; Huang, Bormin

2010-08-01

Low-density parity-check (LDPC) code is a linear block code known to approach the Shannon limit via the iterative sum-product algorithm. LDPC codes have been adopted in most current communication systems such as DVB-S2, WiMAX, WI-FI and 10GBASE-T. LDPC for the needs of reliable and flexible communication links for a wide variety of communication standards and configurations have inspired the demand for high-performance and flexibility computing. Accordingly, finding a fast and reconfigurable developing platform for designing the high-throughput LDPC decoder has become important especially for rapidly changing communication standards and configurations. In this paper, a new graphic-processing-unit (GPU) LDPC decoding platform with the asynchronous data transfer is proposed to realize this practical implementation. Experimental results showed that the proposed GPU-based decoder achieved 271x speedup compared to its CPU-based counterpart. It can serve as a high-throughput LDPC decoder.

High-throughput sequencing of the B-cell receptor in African Burkitt lymphoma reveals clues to pathogenesis.

Science.gov (United States)

Lombardo, Katharine A; Coffey, David G; Morales, Alicia J; Carlson, Christopher S; Towlerton, Andrea M H; Gerdts, Sarah E; Nkrumah, Francis K; Neequaye, Janet; Biggar, Robert J; Orem, Jackson; Casper, Corey; Mbulaiteye, Sam M; Bhatia, Kishor G; Warren, Edus H

2017-03-28

Burkitt lymphoma (BL), the most common pediatric cancer in sub-Saharan Africa, is a malignancy of antigen-experienced B lymphocytes. High-throughput sequencing (HTS) of the immunoglobulin heavy ( IGH ) and light chain ( IGK / IGL ) loci was performed on genomic DNA from 51 primary BL tumors: 19 from Uganda and 32 from Ghana. Reverse transcription polymerase chain reaction analysis and tumor RNA sequencing (RNAseq) was performed on the Ugandan tumors to confirm and extend the findings from the HTS of tumor DNA. Clonal IGH and IGK / IGL rearrangements were identified in 41 and 46 tumors, respectively. Evidence for rearrangement of the second IGH allele was observed in only 6 of 41 tumor samples with a clonal IGH rearrangement, suggesting that the normal process of biallelic IGHD to IGHJ diversity-joining (DJ) rearrangement is often disrupted in BL progenitor cells. Most tumors, including those with a sole dominant, nonexpressed DJ rearrangement, contained many IGH and IGK / IGL sequences that differed from the dominant rearrangement by < 10 nucleotides, suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells.

Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.

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Wesley Morovic

2016-11-01

Full Text Available Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation well defined strains, encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species, and determined their content using plate counting, targeted genotyping. Additionally, strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS of the 16S rRNA sequence using paired-end 2x250bp Illumina MiSeq technology. Using multiple methods, we tested the hypothesis that products do contain the quantitative amount of labeled bacteria, and qualitative list of labeled microbial species. We found that 17 samples (33% were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58% of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to qualitatively determine the correct taxonomy and quantitatively ascertain the relative amounts of mixed microbial populations in commercial probiotic products.

High-throughput sequencing of RNA silencing-associated small RNAs in olive (Olea europaea L..

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Livia Donaire

Full Text Available Small RNAs (sRNAs of 20 to 25 nucleotides (nt in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.. sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive.

Universal and blocking primer mismatches limit the use of high-throughput DNA sequencing for the quantitative metabarcoding of arthropods.

Science.gov (United States)

Piñol, J; Mir, G; Gomez-Polo, P; Agustí, N

2015-07-01

The quantification of the biological diversity in environmental samples using high-throughput DNA sequencing is hindered by the PCR bias caused by variable primer-template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator-specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high-throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer-template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer-template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region. © 2014 John Wiley & Sons Ltd.

Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

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Santos Carla S

2012-11-01

Full Text Available Abstract Background Pine wilt disease (PWD, caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus, damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN and Pinus pinea (less susceptible to PWN. Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species

Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

Science.gov (United States)

2012-01-01

Background Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN). Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species. Conclusions Defense-related genes

Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing

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Zhewei Song

2017-07-01

Full Text Available Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces and lactic acid bacteria (genus Lactobacillus classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol to acid (lactic acid and acetic acid in Chinese Maotai-flavor liquor production. Our findings provide

Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing.

Science.gov (United States)

Song, Zhewei; Du, Hai; Zhang, Yan; Xu, Yan

2017-01-01

Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces , and Zygosaccharomyces ) and lactic acid bacteria (genus Lactobacillus ) classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol) production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic acid) in Chinese Maotai-flavor liquor production. Our findings provide insight into

High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China.

Science.gov (United States)

Wei, Yu-Jie; Wu, Yun; Yan, Yin-Zhuo; Zou, Wan; Xue, Jie; Ma, Wen-Rui; Wang, Wei; Tian, Ge; Wang, Li-Ye

2018-01-01

In this study Illumina MiSeq was performed to investigate microbial diversity in soil, leaves, grape, grape juice and wine. A total of 1,043,102 fungal Internal Transcribed Spacer (ITS) reads and 2,422,188 high quality bacterial 16S rDNA sequences were used for taxonomic classification, revealed five fungal and eight bacterial phyla. At the genus level, the dominant fungi were Ascomycota, Sordariales, Tetracladium and Geomyces in soil, Aureobasidium and Pleosporaceae in grapes leaves, Aureobasidium in grape and grape juice. The dominant bacteria were Kaistobacter, Arthrobacter, Skermanella and Sphingomonas in soil, Pseudomonas, Acinetobacter and Kaistobacter in grape and grapes leaves, and Oenococcus in grape juice and wine. Principal coordinate analysis showed structural separation between the composition of fungi and bacteria in all samples. This is the first study to understand microbiome population in soil, grape, grapes leaves, grape juice and wine in Xinjiang through High-throughput Sequencing and identify microorganisms like Saccharomyces cerevisiae and Oenococcus spp. that may contribute to the quality and flavor of wine.

High-throughput sequencing of microbial community diversity in soil, grapes, leaves, grape juice and wine of grapevine from China

Science.gov (United States)

Yan, Yin-zhuo; Zou, Wan; Ma, Wen-rui; Wang, Wei; Tian, Ge; Wang, Li-ye

2018-01-01

In this study Illumina MiSeq was performed to investigate microbial diversity in soil, leaves, grape, grape juice and wine. A total of 1,043,102 fungal Internal Transcribed Spacer (ITS) reads and 2,422,188 high quality bacterial 16S rDNA sequences were used for taxonomic classification, revealed five fungal and eight bacterial phyla. At the genus level, the dominant fungi were Ascomycota, Sordariales, Tetracladium and Geomyces in soil, Aureobasidium and Pleosporaceae in grapes leaves, Aureobasidium in grape and grape juice. The dominant bacteria were Kaistobacter, Arthrobacter, Skermanella and Sphingomonas in soil, Pseudomonas, Acinetobacter and Kaistobacter in grape and grapes leaves, and Oenococcus in grape juice and wine. Principal coordinate analysis showed structural separation between the composition of fungi and bacteria in all samples. This is the first study to understand microbiome population in soil, grape, grapes leaves, grape juice and wine in Xinjiang through High-throughput Sequencing and identify microorganisms like Saccharomyces cerevisiae and Oenococcus spp. that may contribute to the quality and flavor of wine. PMID:29565999

Fungi Sailing the Arctic Ocean: Speciose Communities in North Atlantic Driftwood as Revealed by High-Throughput Amplicon Sequencing.

Science.gov (United States)

Rämä, Teppo; Davey, Marie L; Nordén, Jenni; Halvorsen, Rune; Blaalid, Rakel; Mathiassen, Geir H; Alsos, Inger G; Kauserud, Håvard

2016-08-01

High amounts of driftwood sail across the oceans and provide habitat for organisms tolerating the rough and saline environment. Fungi have adapted to the extremely cold and saline conditions which driftwood faces in the high north. For the first time, we applied high-throughput sequencing to fungi residing in driftwood to reveal their taxonomic richness, community composition, and ecology in the North Atlantic. Using pyrosequencing of ITS2 amplicons obtained from 49 marine logs, we found 807 fungal operational taxonomic units (OTUs) based on clustering at 97 % sequence similarity cut-off level. The phylum Ascomycota comprised 74 % of the OTUs and 20 % belonged to Basidiomycota. The richness of basidiomycetes decreased with prolonged submersion in the sea, supporting the general view of ascomycetes being more extremotolerant. However, more than one fourth of the fungal OTUs remained unassigned to any fungal class, emphasising the need for better DNA reference data from the marine habitat. Different fungal communities were detected in coniferous and deciduous logs. Our results highlight that driftwood hosts a considerably higher fungal diversity than currently known. The driftwood fungal community is not a terrestrial relic but a speciose assemblage of fungi adapted to the stressful marine environment and different kinds of wooden substrates found in it.

High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

2016-01-01

for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

Applications of High Throughput Nucleotide Sequencing

DEFF Research Database (Denmark)

Waage, Johannes Eichler

equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......-sequencing, a study of the effects on alternative RNA splicing of KO of the nonsense mediated RNA decay system in Mus, using digital gene expression and a custom-built exon-exon junction mapping pipeline is presented (article I). Evolved from this work, a Bioconductor package, spliceR, for classifying alternative...

Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

Science.gov (United States)

Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

2015-09-01

The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing.

Science.gov (United States)

Cui, Li; Zhao, Tingting; Hu, Haibing; Zhang, Wen; Hua, Xiuguo

2017-01-01

Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD) patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by α diversity and principal coordinate analysis (PCoA). Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA) trees were created. Results. After data optimization, an average of 121312 ± 19293 reads in CHD patients and 234372 ± 108725 reads in controls was obtained. Reads corresponding to 38 phyla, 90 classes, and 584 genera were detected in CHD patients, whereas 40 phyla, 99 classes, and 775 genera were detected in controls. The proportion of phylum Bacteroidetes (56.12%) was lower and that of phylum Firmicutes was higher (37.06%) in CHD patients than those in the controls (60.92% and 32.06%, P UPGMA tree analysis showed that there were significant differences of gut microbial compositions between the two groups. Conclusion. The diversity and compositions of gut flora were different between CHD patients and healthy controls. The incidence of CHD might be associated with the alteration of gut microbiota.

Association Study of Gut Flora in Coronary Heart Disease through High-Throughput Sequencing

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Li Cui

2017-01-01

Full Text Available Objectives. We aimed to explore the impact of gut microbiota in coronary heart disease (CHD patients through high-throughput sequencing. Methods. A total of 29 CHD in-hospital patients and 35 healthy volunteers as controls were included. Nucleic acids were extracted from fecal samples, followed by α diversity and principal coordinate analysis (PCoA. Based on unweighted UniFrac distance matrices, unweighted-pair group method with arithmetic mean (UPGMA trees were created. Results. After data optimization, an average of 121312±19293 reads in CHD patients and 234372±108725 reads in controls was obtained. Reads corresponding to 38 phyla, 90 classes, and 584 genera were detected in CHD patients, whereas 40 phyla, 99 classes, and 775 genera were detected in controls. The proportion of phylum Bacteroidetes (56.12% was lower and that of phylum Firmicutes was higher (37.06% in CHD patients than those in the controls (60.92% and 32.06%, P<0.05. PCoA and UPGMA tree analysis showed that there were significant differences of gut microbial compositions between the two groups. Conclusion. The diversity and compositions of gut flora were different between CHD patients and healthy controls. The incidence of CHD might be associated with the alteration of gut microbiota.

High-throughput continuous cryopump

International Nuclear Information System (INIS)

Foster, C.A.

1986-01-01

A cryopump with a unique method of regeneration which allows continuous operation at high throughput has been constructed and tested. Deuterium was pumped continuously at a throughput of 30 Torr.L/s at a speed of 2000 L/s and a compression ratio of 200. Argon was pumped at a throughput of 60 Torr.L/s at a speed of 1275 L/s. To produce continuous operation of the pump, a method of regeneration that does not thermally cycle the pump is employed. A small chamber (the ''snail'') passes over the pumping surface and removes the frost from it either by mechanical action with a scraper or by local heating. The material removed is topologically in a secondary vacuum system with low conductance into the primary vacuum; thus, the exhaust can be pumped at pressures up to an effective compression ratio determined by the ratio of the pumping speed to the leakage conductance of the snail. The pump, which is all-metal-sealed and dry and which regenerates every 60 s, would be an ideal system for pumping tritium. Potential fusion applications are for mpmp limiters, for repeating pneumatic pellet injection lines, and for the centrifuge pellet injector spin tank, all of which will require pumping tritium at high throughput. Industrial applications requiring ultraclean pumping of corrosive gases at high throughput, such as the reactive ion etch semiconductor process, may also be feasible

Fabrication of metallic nanoparticles by spinodal dewetting of thin films: A high-throughput approach

Energy Technology Data Exchange (ETDEWEB)

Michalak, William D.; Miller, James B. [U.S. Department of Energy, National Energy Technology Laboratory, Pittsburgh, PA 15262 (United States); Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213 (United States); Yolcu, Cem [Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213 (United States); Gellman, Andrew J., E-mail: gellman@cmu.edu [U.S. Department of Energy, National Energy Technology Laboratory, Pittsburgh, PA 15262 (United States); Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213 (United States)

2012-11-01

Metal nanoparticles on structured supports are used in a variety of technological applications including biosensing, energy harvesting, and electronics. In every case, the functions and properties of the metallic nanostructures depend on both their composition and structure (i.e. size, shape, and spatial distribution). Among the challenges to the development of metal nanoparticles for these applications is the characterization of relationships between their structure and their functional properties over multiple structural degrees of freedom spanning a large range of values. In this work, a method for creating a morphological gradient of metal nanoparticles on a substrate is described. The approach, suited for high-throughput fabrication and characterization, is based on spinodal dewetting of a metallic thin film from its substrate. Through control of initial film thickness, anneal temperature, and anneal time, spinodal dewetting results in supported nanoparticles with well-defined and controlled structure. The approach is demonstrated through its application to preparation of Pd nanoparticles on a silicon nitride substrate. The morphologies of the particles were characterized by scanning electron and atomic force microscopies. Free energy-based stability and topological analyses were used to confirm the dewetting mechanism. In addition, the stability theory provides a connection to the thermophysical properties of the resulting nanoparticle array. The dewetting approach is general to any metal/support system and provides an alternative, inexpensive, and robust means to rapidly create metal nanostructures with control of morphology. It shows promise for large scale production of metal nanoparticles structures, as well as understanding basic stability properties of thin metal films. - Highlights: Black-Right-Pointing-Pointer Pd dewetting from SiN occurs by a spinodal dewetting mechanism. Black-Right-Pointing-Pointer Dewetting occurs at temperatures well below the

Fabrication of metallic nanoparticles by spinodal dewetting of thin films: A high-throughput approach

International Nuclear Information System (INIS)

Michalak, William D.; Miller, James B.; Yolcu, Cem; Gellman, Andrew J.

2012-01-01

Metal nanoparticles on structured supports are used in a variety of technological applications including biosensing, energy harvesting, and electronics. In every case, the functions and properties of the metallic nanostructures depend on both their composition and structure (i.e. size, shape, and spatial distribution). Among the challenges to the development of metal nanoparticles for these applications is the characterization of relationships between their structure and their functional properties over multiple structural degrees of freedom spanning a large range of values. In this work, a method for creating a morphological gradient of metal nanoparticles on a substrate is described. The approach, suited for high-throughput fabrication and characterization, is based on spinodal dewetting of a metallic thin film from its substrate. Through control of initial film thickness, anneal temperature, and anneal time, spinodal dewetting results in supported nanoparticles with well-defined and controlled structure. The approach is demonstrated through its application to preparation of Pd nanoparticles on a silicon nitride substrate. The morphologies of the particles were characterized by scanning electron and atomic force microscopies. Free energy-based stability and topological analyses were used to confirm the dewetting mechanism. In addition, the stability theory provides a connection to the thermophysical properties of the resulting nanoparticle array. The dewetting approach is general to any metal/support system and provides an alternative, inexpensive, and robust means to rapidly create metal nanostructures with control of morphology. It shows promise for large scale production of metal nanoparticles structures, as well as understanding basic stability properties of thin metal films. - Highlights: ► Pd dewetting from SiN occurs by a spinodal dewetting mechanism. ► Dewetting occurs at temperatures well below the melting point of Pd. ► Spinodal dewetting allows

Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils.

Directory of Open Access Journals (Sweden)

Zewei Song

Full Text Available Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS. The following treatments were considered: 1 the amount of soil used in DNA extraction, 2 the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation in the extraction procedure, 3 the amount of DNA template used in PCR, and 4 the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.

High-throughput computational methods and software for quantitative trait locus (QTL) mapping

NARCIS (Netherlands)

Arends, Danny

2014-01-01

De afgelopen jaren zijn vele nieuwe technologieen zoals Tiling arrays en High throughput DNA sequencing een belangrijke rol gaan spelen binnen het onderzoeksveld van de systeem genetica. Voor onderzoekers is het extreem belangrijk om te begrijpen dat deze methodes hun manier van werken zullen gaan

High-throughput multiplex HLA-typing by ligase detection reaction (LDR) and universal array (UA) approach.

Science.gov (United States)

Consolandi, Clarissa

2009-01-01

One major goal of genetic research is to understand the role of genetic variation in living systems. In humans, by far the most common type of such variation involves differences in single DNA nucleotides, and is thus termed single nucleotide polymorphism (SNP). The need for improvement in throughput and reliability of traditional techniques makes it necessary to develop new technologies. Thus the past few years have witnessed an extraordinary surge of interest in DNA microarray technology. This new technology offers the first great hope for providing a systematic way to explore the genome. It permits a very rapid analysis of thousands genes for the purpose of gene discovery, sequencing, mapping, expression, and polymorphism detection. We generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. In particular, we set up a universal array approach in combination with a PCR-LDR (polymerase chain reaction-ligation detection reaction) strategy for allele identification in the HLA gene.

High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform

DEFF Research Database (Denmark)

Fordyce, Sarah Louise; Avila Arcos, Maria del Carmen; Rockenbauer, Eszter

2011-01-01

repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method...

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

Science.gov (United States)

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

Comparison of a rational vs. high throughput approach for rapid salt screening and selection.

Science.gov (United States)

Collman, Benjamin M; Miller, Jonathan M; Seadeek, Christopher; Stambek, Julie A; Blackburn, Anthony C

2013-01-01

In recent years, high throughput (HT) screening has become the most widely used approach for early phase salt screening and selection in a drug discovery/development setting. The purpose of this study was to compare a rational approach for salt screening and selection to those results previously generated using a HT approach. The rational approach involved a much smaller number of initial trials (one salt synthesis attempt per counterion) that were selected based on a few strategic solubility determinations of the free form combined with a theoretical analysis of the ideal solvent solubility conditions for salt formation. Salt screening results for sertraline, tamoxifen, and trazodone using the rational approach were compared to those previously generated by HT screening. The rational approach produced similar results to HT screening, including identification of the commercially chosen salt forms, but with a fraction of the crystallization attempts. Moreover, the rational approach provided enough solid from the very initial crystallization of a salt for more thorough and reliable solid-state characterization and thus rapid decision-making. The crystallization techniques used in the rational approach mimic larger-scale process crystallization, allowing smoother technical transfer of the selected salt to the process chemist.

Use of genotyping by sequencing data to develop a high-throughput and multifunctional SNP panel for conservation applications in Pacific lamprey.

Science.gov (United States)

Hess, Jon E; Campbell, Nathan R; Docker, Margaret F; Baker, Cyndi; Jackson, Aaron; Lampman, Ralph; McIlraith, Brian; Moser, Mary L; Statler, David P; Young, William P; Wildbill, Andrew J; Narum, Shawn R

2015-01-01

Next-generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high-throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high-throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are FST outliers, and five of these outliers are localized within genes and significantly associated with geography, run-timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species' range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species. © 2014 John Wiley & Sons Ltd.

High-throughput characterization methods for lithium batteries

Directory of Open Access Journals (Sweden)

Yingchun Lyu

2017-09-01

Full Text Available The development of high-performance lithium ion batteries requires the discovery of new materials and the optimization of key components. By contrast with traditional one-by-one method, high-throughput method can synthesize and characterize a large number of compositionally varying samples, which is able to accelerate the pace of discovery, development and optimization process of materials. Because of rapid progress in thin film and automatic control technologies, thousands of compounds with different compositions could be synthesized rapidly right now, even in a single experiment. However, the lack of rapid or combinatorial characterization technologies to match with high-throughput synthesis methods, limit the application of high-throughput technology. Here, we review a series of representative high-throughput characterization methods used in lithium batteries, including high-throughput structural and electrochemical characterization methods and rapid measuring technologies based on synchrotron light sources.

HTSSIP: An R package for analysis of high throughput sequencing data from nucleic acid stable isotope probing (SIP experiments.

Directory of Open Access Journals (Sweden)

Nicholas D Youngblut

Full Text Available Combining high throughput sequencing with stable isotope probing (HTS-SIP is a powerful method for mapping in situ metabolic processes to thousands of microbial taxa. However, accurately mapping metabolic processes to taxa is complex and challenging. Multiple HTS-SIP data analysis methods have been developed, including high-resolution stable isotope probing (HR-SIP, multi-window high-resolution stable isotope probing (MW-HR-SIP, quantitative stable isotope probing (qSIP, and ΔBD. Currently, there is no publicly available software designed specifically for analyzing HTS-SIP data. To address this shortfall, we have developed the HTSSIP R package, an open-source, cross-platform toolset for conducting HTS-SIP analyses in a straightforward and easily reproducible manner. The HTSSIP package, along with full documentation and examples, is available from CRAN at https://cran.r-project.org/web/packages/HTSSIP/index.html and Github at https://github.com/buckleylab/HTSSIP.

A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology.

Science.gov (United States)

Wang, Jing; Yang, Xue; Chen, Haofeng; Wang, Xuewei; Wang, Xiangyu; Fang, Yi; Jia, Zhenyu; Gao, Jidong

2017-07-11

RNA in formalin-fixed and paraffin-embedded (FFPE) tissues provides large amount of information indicating disease stages, histological tumor types and grades, as well as clinical outcomes. However, Detection of RNA expression levels in formalin-fixed and paraffin-embedded samples is extremely difficult due to poor RNA quality. Here we developed a high-throughput method, Reverse Transcription-Multiple Ligation-dependent Probe Sequencing (RT-MLPSeq), to determine expression levels of multiple transcripts in FFPE samples. By combining Reverse Transcription-Multiple Ligation-dependent Amplification method and next generation sequencing technology, RT-MLPSeq overcomes the limit of probe length in multiplex ligation-dependent probe amplification assay and thus could detect expression levels of transcripts without quantitative limitations. We proved that different RT-MLPSeq probes targeting on the same transcripts have highly consistent results and the starting RNA/cDNA input could be as little as 1 ng. RT-MLPSeq also presented consistent relative RNA levels of selected 13 genes with reverse transcription quantitative PCR. Finally, we demonstrated the application of the new RT-MLPSeq method by measuring the mRNA expression levels of 21 genes which can be used for accurate calculation of the breast cancer recurrence score - an index that has been widely used for managing breast cancer patients.

Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing

Science.gov (United States)

Egge, Elianne Sirnæs; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

2015-01-01

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September–October (autumn) and lowest in April–May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3–5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. PMID:25893259

Towards Prebiotic Catalytic Amyloids Using High Throughput Screening.

Directory of Open Access Journals (Sweden)

Michael P Friedmann

Full Text Available Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-β-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications.

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

Science.gov (United States)

Kumar, Dhananjay; Dutta, Summi; Singh, Dharmendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

2017-01-01

Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30 % of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust

SNP-PHAGE – High throughput SNP discovery pipeline

Directory of Open Access Journals (Sweden)

Cregan Perry B

2006-10-01

Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs as defined here are single base sequence changes or short insertion/deletions between or within individuals of a given species. As a result of their abundance and the availability of high throughput analysis technologies SNP markers have begun to replace other traditional markers such as restriction fragment length polymorphisms (RFLPs, amplified fragment length polymorphisms (AFLPs and simple sequence repeats (SSRs or microsatellite markers for fine mapping and association studies in several species. For SNP discovery from chromatogram data, several bioinformatics programs have to be combined to generate an analysis pipeline. Results have to be stored in a relational database to facilitate interrogation through queries or to generate data for further analyses such as determination of linkage disequilibrium and identification of common haplotypes. Although these tasks are routinely performed by several groups, an integrated open source SNP discovery pipeline that can be easily adapted by new groups interested in SNP marker development is currently unavailable. Results We developed SNP-PHAGE (SNP discovery Pipeline with additional features for identification of common haplotypes within a sequence tagged site (Haplotype Analysis and GenBank (-dbSNP submissions. This tool was applied for analyzing sequence traces from diverse soybean genotypes to discover over 10,000 SNPs. This package was developed on UNIX/Linux platform, written in Perl and uses a MySQL database. Scripts to generate a user-friendly web interface are also provided with common queries for preliminary data analysis. A machine learning tool developed by this group for increasing the efficiency of SNP discovery is integrated as a part of this package as an optional feature. The SNP-PHAGE package is being made available open source at http://bfgl.anri.barc.usda.gov/ML/snp-phage/. Conclusion SNP-PHAGE provides a bioinformatics

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC libraries using High Resolution Melt analysis

Directory of Open Access Journals (Sweden)

Caligari Peter DS

2010-05-01

Full Text Available Abstract Background The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. Results Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR and High Resolution Melt (HRM analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. Conclusions A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

Repurposing a Benchtop Centrifuge for High-Throughput Single-Molecule Force Spectroscopy.

Science.gov (United States)

Yang, Darren; Wong, Wesley P

2018-01-01

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as high cost, low throughput, technical difficulty, and strict infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope (CFM) adapted to a commercial centrifuge enables use by nonspecialists, and integration with DNA nanoswitches facilitates both reliable measurements and repeated molecular interrogation. Here, we provide detailed protocols for constructing the CFM, creating DNA nanoswitch samples, and carrying out single-molecule force measurements.

A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

Energy Technology Data Exchange (ETDEWEB)

Gao, David [Iowa State Univ., Ames, IA (United States)

1999-11-08

Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping. The authors first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a polyvinylpyrrolidone dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results. The authors also developed a new sieving Matrix for DNA separation based on commercially available poly(vinylpyrrolidone) (PVP). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution. Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10 C with a ramp of 0.7 C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system. They further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected with a 96-vial tray. Simultaneous one-color laser-induced fluorescence

New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics.

Directory of Open Access Journals (Sweden)

Tercio Goes

2014-10-01

Full Text Available The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1, Pb03 (PS2 and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis.In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA from a Gateway-adapted cassette (cALf which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach.We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.

miRanalyzer: an update on the detection and analysis of microRNAs in high-throughput sequencing experiments

Science.gov (United States)

Hackenberg, Michael; Rodríguez-Ezpeleta, Naiara; Aransay, Ana M.

2011-01-01

We present a new version of miRanalyzer, a web server and stand-alone tool for the detection of known and prediction of new microRNAs in high-throughput sequencing experiments. The new version has been notably improved regarding speed, scope and available features. Alignments are now based on the ultrafast short-read aligner Bowtie (granting also colour space support, allowing mismatches and improving speed) and 31 genomes, including 6 plant genomes, can now be analysed (previous version contained only 7). Differences between plant and animal microRNAs have been taken into account for the prediction models and differential expression of both, known and predicted microRNAs, between two conditions can be calculated. Additionally, consensus sequences of predicted mature and precursor microRNAs can be obtained from multiple samples, which increases the reliability of the predicted microRNAs. Finally, a stand-alone version of the miRanalyzer that is based on a local and easily customized database is also available; this allows the user to have more control on certain parameters as well as to use specific data such as unpublished assemblies or other libraries that are not available in the web server. miRanalyzer is available at http://bioinfo2.ugr.es/miRanalyzer/miRanalyzer.php. PMID:21515631

Discovery of J chain in African lungfish (Protopterus dolloi, Sarcopterygii using high throughput transcriptome sequencing: implications in mucosal immunity.

Directory of Open Access Journals (Sweden)

Luca Tacchi

Full Text Available J chain is a small polypeptide responsible for immunoglobulin (Ig polymerization and transport of Igs across mucosal surfaces in higher vertebrates. We identified a J chain in dipnoid fish, the African lungfish (Protopterus dolloi by high throughput sequencing of the transcriptome. P. dolloi J chain is 161 aa long and contains six of the eight Cys residues present in mammalian J chain. Phylogenetic studies place the lungfish J chain closer to tetrapod J chain than to the coelacanth or nurse shark sequences. J chain expression occurs in all P. dolloi immune tissues examined and it increases in the gut and kidney in response to an experimental bacterial infection. Double fluorescent in-situ hybridization shows that 88.5% of IgM⁺ cells in the gut co-express J chain, a significantly higher percentage than in the pre-pyloric spleen. Importantly, J chain expression is not restricted to the B-cell compartment since gut epithelial cells also express J chain. These results improve our current view of J chain from a phylogenetic perspective.

Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice

OpenAIRE

Yang, Wanneng; Guo, Zilong; Huang, Chenglong; Duan, Lingfeng; Chen, Guoxing; Jiang, Ni; Fang, Wei; Feng, Hui; Xie, Weibo; Lian, Xingming; Wang, Gongwei; Luo, Qingming; Zhang, Qifa; Liu, Qian; Xiong, Lizhong

2014-01-01

Even as the study of plant genomics rapidly develops through the use of high-throughput sequencing techniques, traditional plant phenotyping lags far behind. Here we develop a high-throughput rice phenotyping facility (HRPF) to monitor 13 traditional agronomic traits and 2 newly defined traits during the rice growth period. Using genome-wide association studies (GWAS) of the 15 traits, we identify 141 associated loci, 25 of which contain known genes such as the Green Revolution semi-dwarf gen...

Tiered High-Throughput Screening Approach to Identify ...

Science.gov (United States)

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing.

Science.gov (United States)

Egge, Elianne Sirnaes; Johannessen, Torill Vik; Andersen, Tom; Eikrem, Wenche; Bittner, Lucie; Larsen, Aud; Sandaa, Ruth-Anne; Edvardsen, Bente

2015-06-01

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters. © 2015 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

PLAN: a web platform for automating high-throughput BLAST searches and for managing and mining results.

Science.gov (United States)

He, Ji; Dai, Xinbin; Zhao, Xuechun

2007-02-09

BLAST searches are widely used for sequence alignment. The search results are commonly adopted for various functional and comparative genomics tasks such as annotating unknown sequences, investigating gene models and comparing two sequence sets. Advances in sequencing technologies pose challenges for high-throughput analysis of large-scale sequence data. A number of programs and hardware solutions exist for efficient BLAST searching, but there is a lack of generic software solutions for mining and personalized management of the results. Systematically reviewing the results and identifying information of interest remains tedious and time-consuming. Personal BLAST Navigator (PLAN) is a versatile web platform that helps users to carry out various personalized pre- and post-BLAST tasks, including: (1) query and target sequence database management, (2) automated high-throughput BLAST searching, (3) indexing and searching of results, (4) filtering results online, (5) managing results of personal interest in favorite categories, (6) automated sequence annotation (such as NCBI NR and ontology-based annotation). PLAN integrates, by default, the Decypher hardware-based BLAST solution provided by Active Motif Inc. with a greatly improved efficiency over conventional BLAST software. BLAST results are visualized by spreadsheets and graphs and are full-text searchable. BLAST results and sequence annotations can be exported, in part or in full, in various formats including Microsoft Excel and FASTA. Sequences and BLAST results are organized in projects, the data publication levels of which are controlled by the registered project owners. In addition, all analytical functions are provided to public users without registration. PLAN has proved a valuable addition to the community for automated high-throughput BLAST searches, and, more importantly, for knowledge discovery, management and sharing based on sequence alignment results. The PLAN web interface is platform

PLAN: a web platform for automating high-throughput BLAST searches and for managing and mining results

Directory of Open Access Journals (Sweden)

Zhao Xuechun

2007-02-01

Full Text Available Abstract Background BLAST searches are widely used for sequence alignment. The search results are commonly adopted for various functional and comparative genomics tasks such as annotating unknown sequences, investigating gene models and comparing two sequence sets. Advances in sequencing technologies pose challenges for high-throughput analysis of large-scale sequence data. A number of programs and hardware solutions exist for efficient BLAST searching, but there is a lack of generic software solutions for mining and personalized management of the results. Systematically reviewing the results and identifying information of interest remains tedious and time-consuming. Results Personal BLAST Navigator (PLAN is a versatile web platform that helps users to carry out various personalized pre- and post-BLAST tasks, including: (1 query and target sequence database management, (2 automated high-throughput BLAST searching, (3 indexing and searching of results, (4 filtering results online, (5 managing results of personal interest in favorite categories, (6 automated sequence annotation (such as NCBI NR and ontology-based annotation. PLAN integrates, by default, the Decypher hardware-based BLAST solution provided by Active Motif Inc. with a greatly improved efficiency over conventional BLAST software. BLAST results are visualized by spreadsheets and graphs and are full-text searchable. BLAST results and sequence annotations can be exported, in part or in full, in various formats including Microsoft Excel and FASTA. Sequences and BLAST results are organized in projects, the data publication levels of which are controlled by the registered project owners. In addition, all analytical functions are provided to public users without registration. Conclusion PLAN has proved a valuable addition to the community for automated high-throughput BLAST searches, and, more importantly, for knowledge discovery, management and sharing based on sequence alignment results

Opera: reconstructing optimal genomic scaffolds with high-throughput paired-end sequences.

Science.gov (United States)

Gao, Song; Sung, Wing-Kin; Nagarajan, Niranjan

2011-11-01

Scaffolding, the problem of ordering and orienting contigs, typically using paired-end reads, is a crucial step in the assembly of high-quality draft genomes. Even as sequencing technologies and mate-pair protocols have improved significantly, scaffolding programs still rely on heuristics, with no guarantees on the quality of the solution. In this work, we explored the feasibility of an exact solution for scaffolding and present a first tractable solution for this problem (Opera). We also describe a graph contraction procedure that allows the solution to scale to large scaffolding problems and demonstrate this by scaffolding several large real and synthetic datasets. In comparisons with existing scaffolders, Opera simultaneously produced longer and more accurate scaffolds demonstrating the utility of an exact approach. Opera also incorporates an exact quadratic programming formulation to precisely compute gap sizes (Availability: http://sourceforge.net/projects/operasf/ ).

Apple ring rot-responsive putative microRNAs revealed by high-throughput sequencing in Malus × domestica Borkh.

Science.gov (United States)

Yu, Xin-Yi; Du, Bei-Bei; Gao, Zhi-Hong; Zhang, Shi-Jie; Tu, Xu-Tong; Chen, Xiao-Yun; Zhang, Zhen; Qu, Shen-Chun

2014-08-01

MicroRNAs (miRNAs) are small non-coding RNAs, which silence target mRNA via cleavage or translational inhibition to function in regulating gene expression. MiRNAs act as important regulators of plant development and stress response. For understanding the role of miRNAs responsive to apple ring rot stress, we identified disease-responsive miRNAs using high-throughput sequencing in Malus × domestica Borkh.. Four small RNA libraries were constructed from two control strains in M. domestica, crabapple (CKHu) and Fuji Naga-fu No. 6 (CKFu), and two disease stress strains, crabapple (DSHu) and Fuji Naga-fu No. 6 (DSFu). A total of 59 miRNA families were identified and five miRNAs might be responsive to apple ring rot infection and validated via qRT-PCR. Furthermore, we predicted 76 target genes which were regulated by conserved miRNAs potentially. Our study demonstrated that miRNAs was responsive to apple ring rot infection and may have important implications on apple disease resistance.

Bacterial community compositions of coking wastewater treatment plants in steel industry revealed by Illumina high-throughput sequencing.

Science.gov (United States)

Ma, Qiao; Qu, Yuanyuan; Shen, Wenli; Zhang, Zhaojing; Wang, Jingwei; Liu, Ziyan; Li, Duanxing; Li, Huijie; Zhou, Jiti

2015-03-01

In this study, Illumina high-throughput sequencing was used to reveal the community structures of nine coking wastewater treatment plants (CWWTPs) in China for the first time. The sludge systems exhibited a similar community composition at each taxonomic level. Compared to previous studies, some of the core genera in municipal wastewater treatment plants such as Zoogloea, Prosthecobacter and Gp6 were detected as minor species. Thiobacillus (20.83%), Comamonas (6.58%), Thauera (4.02%), Azoarcus (7.78%) and Rhodoplanes (1.42%) were the dominant genera shared by at least six CWWTPs. The percentages of autotrophic ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were unexpectedly low, which were verified by both real-time PCR and fluorescence in situ hybridization analyses. Hierarchical clustering and canonical correspondence analysis indicated that operation mode, flow rate and temperature might be the key factors in community formation. This study provides new insights into our understanding of microbial community compositions and structures of CWWTPs. Copyright © 2014 Elsevier Ltd. All rights reserved.

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

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Guangbo Liu

Full Text Available Saccharomyces cerevisiae (budding yeast is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids or genome mutation (e.g., gene mutation, deletion, epitope tagging is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

Statistical Approaches for Next-Generation Sequencing Data

OpenAIRE

Qiao, Dandi

2012-01-01

During the last two decades, genotyping technology has advanced rapidly, which enabled the tremendous success of genome-wide association studies (GWAS) in the search of disease susceptibility loci (DSLs). However, only a small fraction of the overall predicted heritability can be explained by the DSLs discovered. One possible explanation for this ”missing heritability” phenomenon is that many causal variants are rare. The recent development of high-throughput next-generation sequencing (NGS) ...

Cancer panomics: computational methods and infrastructure for integrative analysis of cancer high-throughput "omics" data

DEFF Research Database (Denmark)

Brunak, Søren; De La Vega, Francisco M.; Rätsch, Gunnar

2014-01-01

Targeted cancer treatment is becoming the goal of newly developed oncology medicines and has already shown promise in some spectacular cases such as the case of BRAF kinase inhibitors in BRAF-mutant (e.g. V600E) melanoma. These developments are driven by the advent of high-throughput sequencing......, which continues to drop in cost, and that has enabled the sequencing of the genome, transcriptome, and epigenome of the tumors of a large number of cancer patients in order to discover the molecular aberrations that drive the oncogenesis of several types of cancer. Applying these technologies...... in the clinic promises to transform cancer treatment by identifying therapeutic vulnerabilities of each patient's tumor. These approaches will need to address the panomics of cancer--the integration of the complex combination of patient-specific characteristics that drive the development of each person's tumor...

A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform.

Science.gov (United States)

de Muinck, Eric J; Trosvik, Pål; Gilfillan, Gregor D; Hov, Johannes R; Sundaram, Arvind Y M

2017-07-06

Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized. We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms. The library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost. Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length

Identification and characterization of microRNAs related to salt stress in broccoli, using high-throughput sequencing and bioinformatics analysis.

Science.gov (United States)

Tian, Yunhong; Tian, Yunming; Luo, Xiaojun; Zhou, Tao; Huang, Zuoping; Liu, Ying; Qiu, Yihan; Hou, Bing; Sun, Dan; Deng, Hongyu; Qian, Shen; Yao, Kaitai

2014-09-03

MicroRNAs (miRNAs) are a new class of endogenous regulators of a broad range of physiological processes, which act by regulating gene expression post-transcriptionally. The brassica vegetable, broccoli (Brassica oleracea var. italica), is very popular with a wide range of consumers, but environmental stresses such as salinity are a problem worldwide in restricting its growth and yield. Little is known about the role of miRNAs in the response of broccoli to salt stress. In this study, broccoli subjected to salt stress and broccoli grown under control conditions were analyzed by high-throughput sequencing. Differential miRNA expression was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). The prediction of miRNA targets was undertaken using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) database and Gene Ontology (GO)-enrichment analyses. Two libraries of small (or short) RNAs (sRNAs) were constructed and sequenced by high-throughput Solexa sequencing. A total of 24,511,963 and 21,034,728 clean reads, representing 9,861,236 (40.23%) and 8,574,665 (40.76%) unique reads, were obtained for control and salt-stressed broccoli, respectively. Furthermore, 42 putative known and 39 putative candidate miRNAs that were differentially expressed between control and salt-stressed broccoli were revealed by their read counts and confirmed by the use of stem-loop real-time RT-PCR. Amongst these, the putative conserved miRNAs, miR393 and miR855, and two putative candidate miRNAs, miR3 and miR34, were the most strongly down-regulated when broccoli was salt-stressed, whereas the putative conserved miRNA, miR396a, and the putative candidate miRNA, miR37, were the most up-regulated. Finally, analysis of the predicted gene targets of miRNAs using the GO and KO databases indicated that a range of metabolic and other cellular functions known to be associated with salt stress were up-regulated in broccoli treated with salt. A comprehensive

High-throughput electrical characterization for robust overlay lithography control

Science.gov (United States)

Devender, Devender; Shen, Xumin; Duggan, Mark; Singh, Sunil; Rullan, Jonathan; Choo, Jae; Mehta, Sohan; Tang, Teck Jung; Reidy, Sean; Holt, Jonathan; Kim, Hyung Woo; Fox, Robert; Sohn, D. K.

2017-03-01

Realizing sensitive, high throughput and robust overlay measurement is a challenge in current 14nm and advanced upcoming nodes with transition to 300mm and upcoming 450mm semiconductor manufacturing, where slight deviation in overlay has significant impact on reliability and yield1). Exponentially increasing number of critical masks in multi-patterning lithoetch, litho-etch (LELE) and subsequent LELELE semiconductor processes require even tighter overlay specification2). Here, we discuss limitations of current image- and diffraction- based overlay measurement techniques to meet these stringent processing requirements due to sensitivity, throughput and low contrast3). We demonstrate a new electrical measurement based technique where resistance is measured for a macro with intentional misalignment between two layers. Overlay is quantified by a parabolic fitting model to resistance where minima and inflection points are extracted to characterize overlay control and process window, respectively. Analyses using transmission electron microscopy show good correlation between actual overlay performance and overlay obtained from fitting. Additionally, excellent correlation of overlay from electrical measurements to existing image- and diffraction- based techniques is found. We also discuss challenges of integrating electrical measurement based approach in semiconductor manufacturing from Back End of Line (BEOL) perspective. Our findings open up a new pathway for accessing simultaneous overlay as well as process window and margins from a robust, high throughput and electrical measurement approach.

High throughput sample processing and automated scoring

Directory of Open Access Journals (Sweden)

Gunnar eBrunborg

2014-10-01

Full Text Available The comet assay is a sensitive and versatile method for assessing DNA damage in cells. In the traditional version of the assay, there are many manual steps involved and few samples can be treated in one experiment. High throughput modifications have been developed during recent years, and they are reviewed and discussed. These modifications include accelerated scoring of comets; other important elements that have been studied and adapted to high throughput are cultivation and manipulation of cells or tissues before and after exposure, and freezing of treated samples until comet analysis and scoring. High throughput methods save time and money but they are useful also for other reasons: large-scale experiments may be performed which are otherwise not practicable (e.g., analysis of many organs from exposed animals, and human biomonitoring studies, and automation gives more uniform sample treatment and less dependence on operator performance. The high throughput modifications now available vary largely in their versatility, capacity, complexity and costs. The bottleneck for further increase of throughput appears to be the scoring.

SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

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Velasco Riccardo

2008-01-01

Full Text Available Abstract Background Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP discovery and genotyping in grapevine (Vitis vinifera L.. However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs thus providing a valuable source for high-throughput genotyping methods. Results Herein we report the first application of the SNPlex™ genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA methods were used for preparation of genomic DNA for the SNPlex assay. Conclusion Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA, is a good solution for future applications in well-equipped laboratories.

[Research on soil bacteria under the impact of sealed CO2 leakage by high-throughput sequencing technology].

Science.gov (United States)

Tian, Di; Ma, Xin; Li, Yu-E; Zha, Liang-Song; Wu, Yang; Zou, Xiao-Xia; Liu, Shuang

2013-10-01

Carbon dioxide Capture and Storage has provided a new option for mitigating global anthropogenic CO2 emission with its unique advantages. However, there is a risk of the sealed CO2 leakage, bringing a serious threat to the ecology system. It is widely known that soil microorganisms are closely related to soil health, while the study on the impact of sequestered CO2 leakage on soil microorganisms is quite deficient. In this study, the leakage scenarios of sealed CO2 were constructed and the 16S rRNA genes of soil bacteria were sequenced by Illumina high-throughput sequencing technology on Miseq platform, and related biological analysis was conducted to explore the changes of soil bacterial abundance, diversity and structure. There were 486,645 reads for 43,017 OTUs of 15 soil samples and the results of biological analysis showed that there were differences in the abundance, diversity and community structure of soil bacterial community under different CO, leakage scenarios while the abundance and diversity of the bacterial community declined with the amplification of CO2 leakage quantity and leakage time, and some bacteria species became the dominant bacteria species in the bacteria community, therefore the increase of Acidobacteria species would be a biological indicator for the impact of sealed CO2 leakage on soil ecology system.

High-throughput fragment screening by affinity LC-MS.

Science.gov (United States)

Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

2013-02-01

Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

GxGrare: gene-gene interaction analysis method for rare variants from high-throughput sequencing data.

Science.gov (United States)

Kwon, Minseok; Leem, Sangseob; Yoon, Joon; Park, Taesung

2018-03-19

With the rapid advancement of array-based genotyping techniques, genome-wide association studies (GWAS) have successfully identified common genetic variants associated with common complex diseases. However, it has been shown that only a small proportion of the genetic etiology of complex diseases could be explained by the genetic factors identified from GWAS. This missing heritability could possibly be explained by gene-gene interaction (epistasis) and rare variants. There has been an exponential growth of gene-gene interaction analysis for common variants in terms of methodological developments and practical applications. Also, the recent advancement of high-throughput sequencing technologies makes it possible to conduct rare variant analysis. However, little progress has been made in gene-gene interaction analysis for rare variants. Here, we propose GxGrare which is a new gene-gene interaction method for the rare variants in the framework of the multifactor dimensionality reduction (MDR) analysis. The proposed method consists of three steps; 1) collapsing the rare variants, 2) MDR analysis for the collapsed rare variants, and 3) detect top candidate interaction pairs. GxGrare can be used for the detection of not only gene-gene interactions, but also interactions within a single gene. The proposed method is illustrated with 1080 whole exome sequencing data of the Korean population in order to identify causal gene-gene interaction for rare variants for type 2 diabetes. The proposed GxGrare performs well for gene-gene interaction detection with collapsing of rare variants. GxGrare is available at http://bibs.snu.ac.kr/software/gxgrare which contains simulation data and documentation. Supported operating systems include Linux and OS X.

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

Science.gov (United States)

Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

2018-01-16

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice

Science.gov (United States)

Yang, Wanneng; Guo, Zilong; Huang, Chenglong; Duan, Lingfeng; Chen, Guoxing; Jiang, Ni; Fang, Wei; Feng, Hui; Xie, Weibo; Lian, Xingming; Wang, Gongwei; Luo, Qingming; Zhang, Qifa; Liu, Qian; Xiong, Lizhong

2014-01-01

Even as the study of plant genomics rapidly develops through the use of high-throughput sequencing techniques, traditional plant phenotyping lags far behind. Here we develop a high-throughput rice phenotyping facility (HRPF) to monitor 13 traditional agronomic traits and 2 newly defined traits during the rice growth period. Using genome-wide association studies (GWAS) of the 15 traits, we identify 141 associated loci, 25 of which contain known genes such as the Green Revolution semi-dwarf gene, SD1. Based on a performance evaluation of the HRPF and GWAS results, we demonstrate that high-throughput phenotyping has the potential to replace traditional phenotyping techniques and can provide valuable gene identification information. The combination of the multifunctional phenotyping tools HRPF and GWAS provides deep insights into the genetic architecture of important traits. PMID:25295980

High Throughput Neuro-Imaging Informatics

Directory of Open Access Journals (Sweden)

Michael I Miller

2013-12-01

Full Text Available This paper describes neuroinformatics technologies at 1 mm anatomical scale based on high throughput 3D functional and structural imaging technologies of the human brain. The core is an abstract pipeline for converting functional and structural imagery into their high dimensional neuroinformatic representations index containing O(E3-E4 discriminating dimensions. The pipeline is based on advanced image analysis coupled to digital knowledge representations in the form of dense atlases of the human brain at gross anatomical scale. We demonstrate the integration of these high-dimensional representations with machine learning methods, which have become the mainstay of other fields of science including genomics as well as social networks. Such high throughput facilities have the potential to alter the way medical images are stored and utilized in radiological workflows. The neuroinformatics pipeline is used to examine cross-sectional and personalized analyses of neuropsychiatric illnesses in clinical applications as well as longitudinal studies. We demonstrate the use of high throughput machine learning methods for supporting (i cross-sectional image analysis to evaluate the health status of individual subjects with respect to the population data, (ii integration of image and non-image information for diagnosis and prognosis.

Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

Directory of Open Access Journals (Sweden)

Mari eNyyssönen

2013-09-01

Full Text Available Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 % cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

High Throughput Computing Impact on Meta Genomics (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

Energy Technology Data Exchange (ETDEWEB)

Gore, Brooklin

2011-10-12

This presentation includes a brief background on High Throughput Computing, correlating gene transcription factors, optical mapping, genotype to phenotype mapping via QTL analysis, and current work on next gen sequencing.

A high-throughput pipeline for the design of real-time PCR signatures

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Reifman Jaques

2010-06-01

Full Text Available Abstract Background Pathogen diagnostic assays based on polymerase chain reaction (PCR technology provide high sensitivity and specificity. However, the design of these diagnostic assays is computationally intensive, requiring high-throughput methods to identify unique PCR signatures in the presence of an ever increasing availability of sequenced genomes. Results We present the Tool for PCR Signature Identification (TOPSI, a high-performance computing pipeline for the design of PCR-based pathogen diagnostic assays. The TOPSI pipeline efficiently designs PCR signatures common to multiple bacterial genomes by obtaining the shared regions through pairwise alignments between the input genomes. TOPSI successfully designed PCR signatures common to 18 Staphylococcus aureus genomes in less than 14 hours using 98 cores on a high-performance computing system. Conclusions TOPSI is a computationally efficient, fully integrated tool for high-throughput design of PCR signatures common to multiple bacterial genomes. TOPSI is freely available for download at http://www.bhsai.org/downloads/topsi.tar.gz.

Fractal-like Distributions over the Rational Numbers in High-throughput Biological and Clinical Data

Science.gov (United States)

Trifonov, Vladimir; Pasqualucci, Laura; Dalla-Favera, Riccardo; Rabadan, Raul

2011-12-01

Recent developments in extracting and processing biological and clinical data are allowing quantitative approaches to studying living systems. High-throughput sequencing (HTS), expression profiles, proteomics, and electronic health records (EHR) are some examples of such technologies. Extracting meaningful information from those technologies requires careful analysis of the large volumes of data they produce. In this note, we present a set of fractal-like distributions that commonly appear in the analysis of such data. The first set of examples are drawn from a HTS experiment. Here, the distributions appear as part of the evaluation of the error rate of the sequencing and the identification of tumorogenic genomic alterations. The other examples are obtained from risk factor evaluation and analysis of relative disease prevalence and co-mordbidity as these appear in EHR. The distributions are also relevant to identification of subclonal populations in tumors and the study of quasi-species and intrahost diversity of viral populations.

The French press: a repeatable and high-throughput approach to exercising zebrafish (Danio rerio).

Science.gov (United States)

Usui, Takuji; Noble, Daniel W A; O'Dea, Rose E; Fangmeier, Melissa L; Lagisz, Malgorzata; Hesselson, Daniel; Nakagawa, Shinichi

2018-01-01

Zebrafish are increasingly used as a vertebrate model organism for various traits including swimming performance, obesity and metabolism, necessitating high-throughput protocols to generate standardized phenotypic information. Here, we propose a novel and cost-effective method for exercising zebrafish, using a coffee plunger and magnetic stirrer. To demonstrate the use of this method, we conducted a pilot experiment to show that this simple system provides repeatable estimates of maximal swim performance (intra-class correlation [ICC] = 0.34-0.41) and observe that exercise training of zebrafish on this system significantly increases their maximum swimming speed. We propose this high-throughput and reproducible system as an alternative to traditional linear chamber systems for exercising zebrafish and similarly sized fishes.

Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti.

Science.gov (United States)

Cai, Yingying; Xia, Miaomiao; Dong, Huina; Qian, Yuan; Zhang, Tongcun; Zhu, Beiwei; Wu, Jinchuan; Zhang, Dawei

2018-05-11

As a very important coenzyme in the cell metabolism, Vitamin B 12 (cobalamin, VB 12 ) has been widely used in food and medicine fields. The complete biosynthesis of VB 12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB 12 production. High-yield VB 12 -producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. By the help of engineered strains with varied capacities of VB 12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5-2 was obtained and considered as a candidate for industrial applications. After 7 d's cultivation on a rotary shaker at 30 °C, the VB 12 titer of S. meliloti MC5-2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5-2 was sequenced, and gene mutations were identified and analyzed. To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB 12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB 12 -yield strains.

Development of high-throughput SNP-based genotyping in Acacia auriculiformis x A. mangium hybrids using short-read transcriptome data

Directory of Open Access Journals (Sweden)

Wong Melissa ML

2012-12-01

Full Text Available Abstract Background Next Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP discovery in Acacia auriculiformis and Acacia mangium. Like other non-model species, SNP detection and genotyping in Acacia are challenging due to lack of genome sequences. The main objective of this study is to develop the first high-throughput SNP genotyping assay for linkage map construction of A. auriculiformis x A. mangium hybrids. Results We identified a total of 37,786 putative SNPs by aligning short read transcriptome data from four parents of two Acacia hybrid mapping populations using Bowtie against 7,839 de novo transcriptome contigs. Given a set of 10 validated SNPs from two lignin genes, our in silico SNP detection approach is highly accurate (100% compared to the traditional in vitro approach (44%. Further validation of 96 SNPs using Illumina GoldenGate Assay gave an overall assay success rate of 89.6% and conversion rate of 37.5%. We explored possible factors lowering assay success rate by predicting exon-intron boundaries and paralogous genes of Acacia contigs using Medicago truncatula genome as reference. This assessment revealed that presence of exon-intron boundary is the main cause (50% of assay failure. Subsequent SNPs filtering and improved assay design resulted in assay success and conversion rate of 92.4% and 57.4%, respectively based on 768 SNPs genotyping. Analysis of clustering patterns revealed that 27.6% of the assays were not reproducible and flanking sequence might play a role in determining cluster compression. In addition, we identified a total of 258 and 319 polymorphic SNPs in A. auriculiformis and A. mangium natural germplasms, respectively. Conclusion We have successfully discovered a large number of SNP markers in A. auriculiformis x A. mangium hybrids using next generation transcriptome sequencing. By using a reference genome from the most closely

High-Throughput Sequencing of Microbial Community Diversity and Dynamics during Douchi Fermentation

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Tu, Zong-cai; Wang, Xiao-lan

2016-01-01

Douchi is a type of Chinese traditional fermented food that is an important source of protein and is used in flavouring ingredients. The end product is affected by the microbial community present during fermentation, but exactly how microbes influence the fermentation process remains poorly understood. We used an Illumina MiSeq approach to investigate bacterial and fungal community diversity during both douchi-koji making and fermentation. A total of 181,443 high quality bacterial 16S rRNA sequences and 221,059 high quality fungal internal transcribed spacer reads were used for taxonomic classification, revealing eight bacterial and three fungal phyla. Firmicutes, Actinobacteria and Proteobacteria were the dominant bacterial phyla, while Ascomycota and Zygomycota were the dominant fungal phyla. At the genus level, Staphylococcus and Weissella were the dominant bacteria, while Aspergillus and Lichtheimia were the dominant fungi. Principal coordinate analysis showed structural separation between the composition of bacteria in koji making and fermentation. However, multivariate analysis of variance based on unweighted UniFrac distances did identify distinct differences (p fermentation. This is the first investigation to integrate douchi fermentation and koji making and fermentation processes through this technological approach. The results provide insight into the microbiome of the douchi fermentation process, and reveal a structural separation that may be stratified by the environment during the production of this traditional fermented food. PMID:27992473

Infra-red thermography for high throughput field phenotyping in Solanum tuberosum.

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Ankush Prashar

Full Text Available The rapid development of genomic technology has made high throughput genotyping widely accessible but the associated high throughput phenotyping is now the major limiting factor in genetic analysis of traits. This paper evaluates the use of thermal imaging for the high throughput field phenotyping of Solanum tuberosum for differences in stomatal behaviour. A large multi-replicated trial of a potato mapping population was used to investigate the consistency in genotypic rankings across different trials and across measurements made at different times of day and on different days. The results confirmed a high degree of consistency between the genotypic rankings based on relative canopy temperature on different occasions. Genotype discrimination was enhanced both through normalising data by expressing genotype temperatures as differences from image means and through the enhanced replication obtained by using overlapping images. A Monte Carlo simulation approach was used to confirm the magnitude of genotypic differences that it is possible to discriminate. The results showed a clear negative association between canopy temperature and final tuber yield for this population, when grown under ample moisture supply. We have therefore established infrared thermography as an easy, rapid and non-destructive screening method for evaluating large population trials for genetic analysis. We also envisage this approach as having great potential for evaluating plant response to stress under field conditions.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

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Jason D Thompson

Full Text Available Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

Science.gov (United States)

Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

MetaGenSense: A web-application for analysis and exploration of high throughput sequencing metagenomic data [version 3; referees: 1 approved, 2 approved with reservations

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Damien Correia

2016-12-01

Full Text Available The detection and characterization of emerging infectious agents has been a continuing public health concern. High Throughput Sequencing (HTS or Next-Generation Sequencing (NGS technologies have proven to be promising approaches for efficient and unbiased detection of pathogens in complex biological samples, providing access to comprehensive analyses. As NGS approaches typically yield millions of putatively representative reads per sample, efficient data management and visualization resources have become mandatory. Most usually, those resources are implemented through a dedicated Laboratory Information Management System (LIMS, solely to provide perspective regarding the available information. We developed an easily deployable web-interface, facilitating management and bioinformatics analysis of metagenomics data-samples. It was engineered to run associated and dedicated Galaxy workflows for the detection and eventually classification of pathogens. The web application allows easy interaction with existing Galaxy metagenomic workflows, facilitates the organization, exploration and aggregation of the most relevant sample-specific sequences among millions of genomic sequences, allowing them to determine their relative abundance, and associate them to the most closely related organism or pathogen. The user-friendly Django-Based interface, associates the users’ input data and its metadata through a bio-IT provided set of resources (a Galaxy instance, and both sufficient storage and grid computing power. Galaxy is used to handle and analyze the user’s input data from loading, indexing, mapping, assembly and DB-searches. Interaction between our application and Galaxy is ensured by the BioBlend library, which gives API-based access to Galaxy’s main features. Metadata about samples, runs, as well as the workflow results are stored in the LIMS. For metagenomic classification and exploration purposes, we show, as a proof of concept, that integration

[New-generation high-throughput technologies based 'omics' research strategy in human disease].

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Yang, Xu; Jiao, Rui; Yang, Lin; Wu, Li-Ping; Li, Ying-Rui; Wang, Jun

2011-08-01

In recent years, new-generation high-throughput technologies, including next-generation sequencing technology and mass spectrometry method, have been widely applied in solving biological problems, especially in human diseases field. This data driven, large-scale and industrialized research model enables the omnidirectional and multi-level study of human diseases from the perspectives of genomics, transcriptomics and proteomics levels, etc. In this paper, the latest development of the high-throughput technologies that applied in DNA, RNA, epigenomics, metagenomics including proteomics and some applications in translational medicine are reviewed. At genomics level, exome sequencing has been the hot spot of the recent research. However, the predominance of whole genome resequencing in detecting large structural variants within the whole genome level is coming to stand out as the drop of sequencing cost, which also makes it possible for personalized genome based medicine application. At trancriptomics level, e.g., small RNA sequencing can be used to detect known and predict unknown miRNA. Those small RNA could not only be the biomarkers for disease diagnosis and prognosis, but also show the potential of disease treatment. At proteomics level, e.g., target proteomics can be used to detect the possible disease-related protein or peptides, which can be useful index for clinical staging and typing. Furthermore, the application and development of trans-omics study in disease research are briefly introduced. By applying bioinformatics technologies for integrating multi-omics data, the mechanism, diagnosis and therapy of the disease are likely to be systemically explained and realized, so as to provide powerful tools for disease diagnosis and therapies.

High-throughput search for caloric materials: the CaloriCool approach

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Zarkevich, N. A.; Johnson, D. D.; Pecharsky, V. K.

2018-01-01

The high-throughput search paradigm adopted by the newly established caloric materials consortium—CaloriCool®—with the goal to substantially accelerate discovery and design of novel caloric materials is briefly discussed. We begin with describing material selection criteria based on known properties, which are then followed by heuristic fast estimates, ab initio calculations, all of which has been implemented in a set of automated computational tools and measurements. We also demonstrate how theoretical and computational methods serve as a guide for experimental efforts by considering a representative example from the field of magnetocaloric materials.

Advances in High-Throughput Speed, Low-Latency Communication for Embedded Instrumentation (7th Annual SFAF Meeting, 2012)

Energy Technology Data Exchange (ETDEWEB)

Jordan, Scott

2012-06-01

Scott Jordan on "Advances in high-throughput speed, low-latency communication for embedded instrumentation" at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

A robust, simple genotyping-by-sequencing (GBS approach for high diversity species.

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Robert J Elshire

Full Text Available Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs. This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM and barley (Oregon Wolfe Barley recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

High-throughput sequencing and copy number variation detection using formalin fixed embedded tissue in metastatic gastric cancer.

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Seokhwi Kim

Full Text Available In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%, APC (10.1%, PIK3CA (5.6%, KRAS (4.5%, SMO (3.4%, STK11 (3.4%, CDKN2A (3.4% and SMAD4 (3.4%. Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%, 4 (4.5%, 2 (2.2%, 1 (1.1% and 1 (1.1% cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes.

Novel strategy for protein exploration: high-throughput screening assisted with fuzzy neural network.

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Kato, Ryuji; Nakano, Hideo; Konishi, Hiroyuki; Kato, Katsuya; Koga, Yuchi; Yamane, Tsuneo; Kobayashi, Takeshi; Honda, Hiroyuki

2005-08-19

To engineer proteins with desirable characteristics from a naturally occurring protein, high-throughput screening (HTS) combined with directed evolutional approach is the essential technology. However, most HTS techniques are simple positive screenings. The information obtained from the positive candidates is used only as results but rarely as clues for understanding the structural rules, which may explain the protein activity. In here, we have attempted to establish a novel strategy for exploring functional proteins associated with computational analysis. As a model case, we explored lipases with inverted enantioselectivity for a substrate p-nitrophenyl 3-phenylbutyrate from the wild-type lipase of Burkhorderia cepacia KWI-56, which is originally selective for (S)-configuration of the substrate. Data from our previous work on (R)-enantioselective lipase screening were applied to fuzzy neural network (FNN), bioinformatic algorithm, to extract guidelines for screening and engineering processes to be followed. FNN has an advantageous feature of extracting hidden rules that lie between sequences of variants and their enzyme activity to gain high prediction accuracy. Without any prior knowledge, FNN predicted a rule indicating that "size at position L167," among four positions (L17, F119, L167, and L266) in the substrate binding core region, is the most influential factor for obtaining lipase with inverted (R)-enantioselectivity. Based on the guidelines obtained, newly engineered novel variants, which were not found in the actual screening, were experimentally proven to gain high (R)-enantioselectivity by engineering the size at position L167. We also designed and assayed two novel variants, namely FIGV (L17F, F119I, L167G, and L266V) and FFGI (L17F, L167G, and L266I), which were compatible with the guideline obtained from FNN analysis, and confirmed that these designed lipases could acquire high inverted enantioselectivity. The results have shown that with the aid of

Whole-exome sequencing and high throughput genotyping identified KCNJ11 as the thirteenth MODY gene.

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Bonnefond, Amélie; Philippe, Julien; Durand, Emmanuelle; Dechaume, Aurélie; Huyvaert, Marlène; Montagne, Louise; Marre, Michel; Balkau, Beverley; Fajardy, Isabelle; Vambergue, Anne; Vatin, Vincent; Delplanque, Jérôme; Le Guilcher, David; De Graeve, Franck; Lecoeur, Cécile; Sand, Olivier; Vaxillaire, Martine; Froguel, Philippe

2012-01-01

Maturity-onset of the young (MODY) is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X). Here, we aimed to use whole-exome sequencing (WES) in a four-generation MODY-X family to identify a new susceptibility gene for MODY. WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing) was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay) of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out. By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130) present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11) co-segregated with diabetes in the family (with a LOD-score of 3.68). No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects. Beyond neonatal diabetes mellitus (NDM), KCNJ11 is also a MODY gene ('MODY13'), confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS). Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.

Whole-exome sequencing and high throughput genotyping identified KCNJ11 as the thirteenth MODY gene.

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Amélie Bonnefond

Full Text Available BACKGROUND: Maturity-onset of the young (MODY is a clinically heterogeneous form of diabetes characterized by an autosomal-dominant mode of inheritance, an onset before the age of 25 years, and a primary defect in the pancreatic beta-cell function. Approximately 30% of MODY families remain genetically unexplained (MODY-X. Here, we aimed to use whole-exome sequencing (WES in a four-generation MODY-X family to identify a new susceptibility gene for MODY. METHODOLOGY: WES (Agilent-SureSelect capture/Illumina-GAIIx sequencing was performed in three affected and one non-affected relatives in the MODY-X family. We then performed a high-throughput multiplex genotyping (Illumina-GoldenGate assay of the putative causal mutations in the whole family and in 406 controls. A linkage analysis was also carried out. PRINCIPAL FINDINGS: By focusing on variants of interest (i.e. gains of stop codon, frameshift, non-synonymous and splice-site variants not reported in dbSNP130 present in the three affected relatives and not present in the control, we found 69 mutations. However, as WES was not uniform between samples, a total of 324 mutations had to be assessed in the whole family and in controls. Only one mutation (p.Glu227Lys in KCNJ11 co-segregated with diabetes in the family (with a LOD-score of 3.68. No KCNJ11 mutation was found in 25 other MODY-X unrelated subjects. CONCLUSIONS/SIGNIFICANCE: Beyond neonatal diabetes mellitus (NDM, KCNJ11 is also a MODY gene ('MODY13', confirming the wide spectrum of diabetes related phenotypes due to mutations in NDM genes (i.e. KCNJ11, ABCC8 and INS. Therefore, the molecular diagnosis of MODY should include KCNJ11 as affected carriers can be ideally treated with oral sulfonylureas.

TIMPs of parasitic helminths - a large-scale analysis of high-throughput sequence datasets.

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Cantacessi, Cinzia; Hofmann, Andreas; Pickering, Darren; Navarro, Severine; Mitreva, Makedonka; Loukas, Alex

2013-05-30

Tissue inhibitors of metalloproteases (TIMPs) are a multifunctional family of proteins that orchestrate extracellular matrix turnover, tissue remodelling and other cellular processes. In parasitic helminths, such as hookworms, TIMPs have been proposed to play key roles in the host-parasite interplay, including invasion of and establishment in the vertebrate animal hosts. Currently, knowledge of helminth TIMPs is limited to a small number of studies on canine hookworms, whereas no information is available on the occurrence of TIMPs in other parasitic helminths causing neglected diseases. In the present study, we conducted a large-scale investigation of TIMP proteins of a range of neglected human parasites including the hookworm Necator americanus, the roundworm Ascaris suum, the liver flukes Clonorchis sinensis and Opisthorchis viverrini, as well as the schistosome blood flukes. This entailed mining available transcriptomic and/or genomic sequence datasets for the presence of homologues of known TIMPs, predicting secondary structures of defined protein sequences, systematic phylogenetic analyses and assessment of differential expression of genes encoding putative TIMPs in the developmental stages of A. suum, N. americanus and Schistosoma haematobium which infect the mammalian hosts. A total of 15 protein sequences with high homology to known eukaryotic TIMPs were predicted from the complement of sequence data available for parasitic helminths and subjected to in-depth bioinformatic analyses. Supported by the availability of gene manipulation technologies such as RNA interference and/or transgenesis, this work provides a basis for future functional explorations of helminth TIMPs and, in particular, of their role/s in fundamental biological pathways linked to long-term establishment in the vertebrate hosts, with a view towards the development of novel approaches for the control of neglected helminthiases.

microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach.

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Macchiaroli, Natalia; Cucher, Marcela; Zarowiecki, Magdalena; Maldonado, Lucas; Kamenetzky, Laura; Rosenzvit, Mara Cecilia

2015-02-06

microRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. The particular developmental and metabolic characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we perform a comprehensive analysis of miRNAs in the parasitic cestode Echinococcus canadensis G7, one of the causative agents of the neglected zoonotic disease cystic echinococcosis. Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. For miRNA prediction, miRDeep2 core algorithm was used. The output list of candidate precursors was manually curated to generate a high confidence set of miRNAs. Differential expression analysis of miRNAs between stages or species was estimated with DESeq. Expression levels of selected miRNAs were validated using poly-A RT-qPCR. In this study we used a high-throughput approach and found transcriptional evidence of 37 miRNAs thus expanding the miRNA repertoire of E. canadensis G7. Differential expression analysis showed highly regulated miRNAs between life cycle stages, suggesting a role in maintaining the features of each developmental stage or in the regulation of developmental timing. In this work we characterize conserved and novel Echinococcus miRNAs which represent 30 unique miRNA families. Here we confirmed the remarkable loss of conserved miRNA families in E. canadensis, reflecting their low morphological complexity and high adaptation to parasitism. We performed the first in-depth study profiling of small RNAs in the zoonotic parasite E. canadensis G7. We found that miRNAs are the preponderant small RNA silencing molecules, suggesting that these small RNAs could be an essential mechanism of gene regulation in this species. We also

Fine grained compositional analysis of Port Everglades Inlet microbiome using high throughput DNA sequencing.

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O'Connell, Lauren; Gao, Song; McCorquodale, Donald; Fleisher, Jay; Lopez, Jose V

2018-01-01

Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities ("microbiomes") in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus . However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta

Fine grained compositional analysis of Port Everglades Inlet microbiome using high throughput DNA sequencing

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Lauren O’Connell

2018-05-01

Full Text Available Background Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities (“microbiomes” in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Methods & Results Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus. However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Discussion Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups

Achieving high data throughput in research networks

International Nuclear Information System (INIS)

Matthews, W.; Cottrell, L.

2001-01-01

After less than a year of operation, the BaBar experiment at SLAC has collected almost 100 million particle collision events in a database approaching 165TB. Around 20 TB of data has been exported via the Internet to the BaBar regional center at IN2P3 in Lyon, France, and around 40TB of simulated data has been imported from the Lawrence Livermore National Laboratory (LLNL). BaBar collaborators plan to double data collection each year and export a third of the data to IN2P3. So within a few years the SLAC OC3 (155 Mbps) connection will be fully utilized by file transfer to France alone. Upgrades to infrastructure is essential and detailed understanding of performance issues and the requirements for reliable high throughput transfers is critical. In this talk results from active and passive monitoring and direct measurements of throughput will be reviewed. Methods for achieving the ambitious requirements will be discussed

Achieving High Data Throughput in Research Networks

International Nuclear Information System (INIS)

Matthews, W

2004-01-01

After less than a year of operation, the BaBar experiment at SLAC has collected almost 100 million particle collision events in a database approaching 165TB. Around 20 TB of data has been exported via the Internet to the BaBar regional center at IN2P3 in Lyon, France, and around 40TB of simulated data has been imported from the Lawrence Livermore National Laboratory (LLNL). BaBar collaborators plan to double data collection each year and export a third of the data to IN2P3. So within a few years the SLAC OC3 (155Mbps) connection will be fully utilized by file transfer to France alone. Upgrades to infrastructure is essential and detailed understanding of performance issues and the requirements for reliable high throughput transfers is critical. In this talk results from active and passive monitoring and direct measurements of throughput will be reviewed. Methods for achieving the ambitious requirements will be discussed

High-throughput sequencing of microRNAs in peripheral blood mononuclear cells: identification of potential weight loss biomarkers.

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Fermín I Milagro

Full Text Available INTRODUCTION: MicroRNAs (miRNAs are being increasingly studied in relation to energy metabolism and body composition homeostasis. Indeed, the quantitative analysis of miRNAs expression in different adiposity conditions may contribute to understand the intimate mechanisms participating in body weight control and to find new biomarkers with diagnostic or prognostic value in obesity management. OBJECTIVE: The aim of this study was the search for miRNAs in blood cells whose expression could be used as prognostic biomarkers of weight loss. METHODS: Ten Caucasian obese women were selected among the participants in a weight-loss trial that consisted in following an energy-restricted treatment. Weight loss was considered unsuccessful when 5% (responders. At baseline, total miRNA isolated from peripheral blood mononuclear cells (PBMC was sequenced with SOLiD v4. The miRNA sequencing data were validated by RT-PCR. RESULTS: Differential baseline expression of several miRNAs was found between responders and non-responders. Two miRNAs were up-regulated in the non-responder group (mir-935 and mir-4772 and three others were down-regulated (mir-223, mir-224 and mir-376b. Both mir-935 and mir-4772 showed relevant associations with the magnitude of weight loss, although the expression of other transcripts (mir-874, mir-199b, mir-766, mir-589 and mir-148b also correlated with weight loss. CONCLUSIONS: This research addresses the use of high-throughput sequencing technologies in the search for miRNA expression biomarkers in obesity, by determining the miRNA transcriptome of PBMC. Basal expression of different miRNAs, particularly mir-935 and mir-4772, could be prognostic biomarkers and may forecast the response to a hypocaloric diet.

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature.

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Sergeant, Martin J; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W; Pallen, Mark J

2012-01-01

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

High-sensitivity HLA typing by Saturated Tiling Capture Sequencing (STC-Seq).

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Jiao, Yang; Li, Ran; Wu, Chao; Ding, Yibin; Liu, Yanning; Jia, Danmei; Wang, Lifeng; Xu, Xiang; Zhu, Jing; Zheng, Min; Jia, Junling

2018-01-15

Highly polymorphic human leukocyte antigen (HLA) genes are responsible for fine-tuning the adaptive immune system. High-resolution HLA typing is important for the treatment of autoimmune and infectious diseases. Additionally, it is routinely performed for identifying matched donors in transplantation medicine. Although many HLA typing approaches have been developed, the complexity, low-efficiency and high-cost of current HLA-typing assays limit their application in population-based high-throughput HLA typing for donors, which is required for creating large-scale databases for transplantation and precision medicine. Here, we present a cost-efficient Saturated Tiling Capture Sequencing (STC-Seq) approach to capturing 14 HLA class I and II genes. The highly efficient capture (an approximately 23,000-fold enrichment) of these genes allows for simplified allele calling. Tests on five genes (HLA-A/B/C/DRB1/DQB1) from 31 human samples and 351 datasets using STC-Seq showed results that were 98% consistent with the known two sets of digitals (field1 and field2) genotypes. Additionally, STC can capture genomic DNA fragments longer than 3 kb from HLA loci, making the library compatible with the third-generation sequencing. STC-Seq is a highly accurate and cost-efficient method for HLA typing which can be used to facilitate the establishment of population-based HLA databases for the precision and transplantation medicine.

High-throughput sequencing of microRNA transcriptome and expression assay in the sturgeon, Acipenser schrenckii.

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Lihong Yuan

Full Text Available Sturgeons are considered as living fossils and have very high evolutionary, economical and conservation values. The multiploidy of sturgeon that has been caused by chromosome duplication may lead to the emergence of new microRNAs (miRNAs involved in the ploidy and physiological processes. In the present study, we performed the first sturgeon miRNAs analysis by RNA-seq high-throughput sequencing combined with expression assay of microarray and real-time PCR, and aimed to discover the sturgeon-specific miRNAs, confirm the expressed pattern of miRNAs and illustrate the potential role of miRNAs-targets on sturgeon biological processes. A total of 103 miRNAs were identified, including 58 miRNAs with strongly detected signals (signal >500 and P≤0.01, which were detected by microarray. Real-time PCR assay supported the expression pattern obtained by microarray. Moreover, co-expression of 21 miRNAs in all five tissues and tissue-specific expression of 16 miRNAs implied the crucial and particular function of them in sturgeon physiological processes. Target gene prediction, especially the enriched functional gene groups (369 GO terms and pathways (37 KEGG regulated by 58 miRNAs (P<0.05, illustrated the interaction of miRNAs and putative mRNAs, and also the potential mechanism involved in these biological processes. Our new findings of sturgeon miRNAs expand the public database of transcriptome information for this species, contribute to our understanding of sturgeon biology, and also provide invaluable data that may be applied in sturgeon breeding.

High-throughput sequencing of microRNA transcriptome and expression assay in the sturgeon, Acipenser schrenckii.

Science.gov (United States)

Yuan, Lihong; Zhang, Xiujuan; Li, Linmiao; Jiang, Haiying; Chen, Jinping

2014-01-01

Sturgeons are considered as living fossils and have very high evolutionary, economical and conservation values. The multiploidy of sturgeon that has been caused by chromosome duplication may lead to the emergence of new microRNAs (miRNAs) involved in the ploidy and physiological processes. In the present study, we performed the first sturgeon miRNAs analysis by RNA-seq high-throughput sequencing combined with expression assay of microarray and real-time PCR, and aimed to discover the sturgeon-specific miRNAs, confirm the expressed pattern of miRNAs and illustrate the potential role of miRNAs-targets on sturgeon biological processes. A total of 103 miRNAs were identified, including 58 miRNAs with strongly detected signals (signal >500 and P≤0.01), which were detected by microarray. Real-time PCR assay supported the expression pattern obtained by microarray. Moreover, co-expression of 21 miRNAs in all five tissues and tissue-specific expression of 16 miRNAs implied the crucial and particular function of them in sturgeon physiological processes. Target gene prediction, especially the enriched functional gene groups (369 GO terms) and pathways (37 KEGG) regulated by 58 miRNAs (P<0.05), illustrated the interaction of miRNAs and putative mRNAs, and also the potential mechanism involved in these biological processes. Our new findings of sturgeon miRNAs expand the public database of transcriptome information for this species, contribute to our understanding of sturgeon biology, and also provide invaluable data that may be applied in sturgeon breeding.

Next Generation DNA Sequencing and the Future of Genomic Medicine

OpenAIRE

Anderson, Matthew W.; Schrijver, Iris

2010-01-01

In the years since the first complete human genome sequence was reported, there has been a rapid development of technologies to facilitate high-throughput sequence analysis of DNA (termed “next-generation” sequencing). These novel approaches to DNA sequencing offer the promise of complete genomic analysis at a cost feasible for routine clinical diagnostics. However, the ability to more thoroughly interrogate genomic sequence raises a number of important issues with regard to result interpreta...

Multiple and high-throughput droplet reactions via combination of microsampling technique and microfluidic chip

KAUST Repository

Wu, Jinbo

2012-11-20

Microdroplets offer unique compartments for accommodating a large number of chemical and biological reactions in tiny volume with precise control. A major concern in droplet-based microfluidics is the difficulty to address droplets individually and achieve high throughput at the same time. Here, we have combined an improved cartridge sampling technique with a microfluidic chip to perform droplet screenings and aggressive reaction with minimal (nanoliter-scale) reagent consumption. The droplet composition, distance, volume (nanoliter to subnanoliter scale), number, and sequence could be precisely and digitally programmed through the improved sampling technique, while sample evaporation and cross-contamination are effectively eliminated. Our combined device provides a simple model to utilize multiple droplets for various reactions with low reagent consumption and high throughput. © 2012 American Chemical Society.

High-throughput shotgun lipidomics by quadrupole time-of-flight mass spectrometry

DEFF Research Database (Denmark)

Ståhlman, Marcus; Ejsing, Christer S.; Tarasov, Kirill

2009-01-01

Technological advances in mass spectrometry and meticulous method development have produced several shotgun lipidomic approaches capable of characterizing lipid species by direct analysis of total lipid extracts. Shotgun lipidomics by hybrid quadrupole time-of-flight mass spectrometry allows...... the absolute quantification of hundreds of molecular glycerophospholipid species, glycerolipid species, sphingolipid species and sterol lipids. Future applications in clinical cohort studies demand detailed lipid molecule information and the application of high-throughput lipidomics platforms. In this review...... we describe a novel high-throughput shotgun lipidomic platform based on 96-well robot-assisted lipid extraction, automated sample infusion by mircofluidic-based nanoelectrospray ionization, and quantitative multiple precursor ion scanning analysis on a quadrupole time-of-flight mass spectrometer...

Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench.

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Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

2017-06-01

Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. © 2017 Beckers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

High-throughput sequencing and pathway analysis reveal alteration of the pituitary transcriptome by 17α-ethynylestradiol (EE2) in female coho salmon, Oncorhynchus kisutch

Energy Technology Data Exchange (ETDEWEB)

Harding, Louisa B. [School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195 (United States); Schultz, Irvin R. [Battelle, Marine Sciences Laboratory – Pacific Northwest National Laboratory, 1529 West Sequim Bay Road, Sequim, WA 98382 (United States); Goetz, Giles W. [School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195 (United States); Luckenbach, J. Adam [Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd E, Seattle, WA 98112 (United States); Center for Reproductive Biology, Washington State University, Pullman, WA 98164 (United States); Young, Graham [School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195 (United States); Center for Reproductive Biology, Washington State University, Pullman, WA 98164 (United States); Goetz, Frederick W. [Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Manchester Research Station, P.O. Box 130, Manchester, WA 98353 (United States); Swanson, Penny, E-mail: penny.swanson@noaa.gov [Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd E, Seattle, WA 98112 (United States); Center for Reproductive Biology, Washington State University, Pullman, WA 98164 (United States)

2013-10-15

Highlights: •Studied impacts of ethynylestradiol (EE2) exposure on salmon pituitary transcriptome. •High-throughput sequencing, RNAseq, and pathway analysis were performed. •EE2 altered mRNAs for genes in circadian rhythm, GnRH, and TGFβ signaling pathways. •LH and FSH beta subunit mRNAs were most highly up- and down-regulated by EE2, respectively. •Estrogens may alter processes associated with reproductive timing in salmon. -- Abstract: Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain–pituitary–gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12 ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina{sup ®} sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone β subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone β subunit (−3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-β signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks

High-throughput sequencing and pathway analysis reveal alteration of the pituitary transcriptome by 17α-ethynylestradiol (EE2) in female coho salmon, Oncorhynchus kisutch

International Nuclear Information System (INIS)

Harding, Louisa B.; Schultz, Irvin R.; Goetz, Giles W.; Luckenbach, J. Adam; Young, Graham; Goetz, Frederick W.; Swanson, Penny

2013-01-01

Highlights: •Studied impacts of ethynylestradiol (EE2) exposure on salmon pituitary transcriptome. •High-throughput sequencing, RNAseq, and pathway analysis were performed. •EE2 altered mRNAs for genes in circadian rhythm, GnRH, and TGFβ signaling pathways. •LH and FSH beta subunit mRNAs were most highly up- and down-regulated by EE2, respectively. •Estrogens may alter processes associated with reproductive timing in salmon. -- Abstract: Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain–pituitary–gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12 ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina ® sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone β subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone β subunit (−3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-β signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks

MUSCLE: multiple sequence alignment with high accuracy and high throughput.

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Edgar, Robert C

2004-01-01

We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae.

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Byers, James S; Jarosz, Daniel F

2017-08-08

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrP Sc ) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that >1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

PCR cycles above routine numbers do not compromise high-throughput DNA barcoding results.

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Vierna, J; Doña, J; Vizcaíno, A; Serrano, D; Jovani, R

2017-10-01

High-throughput DNA barcoding has become essential in ecology and evolution, but some technical questions still remain. Increasing the number of PCR cycles above the routine 20-30 cycles is a common practice when working with old-type specimens, which provide little amounts of DNA, or when facing annealing issues with the primers. However, increasing the number of cycles can raise the number of artificial mutations due to polymerase errors. In this work, we sequenced 20 COI libraries in the Illumina MiSeq platform. Libraries were prepared with 40, 45, 50, 55, and 60 PCR cycles from four individuals belonging to four species of four genera of cephalopods. We found no relationship between the number of PCR cycles and the number of mutations despite using a nonproofreading polymerase. Moreover, even when using a high number of PCR cycles, the resulting number of mutations was low enough not to be an issue in the context of high-throughput DNA barcoding (but may still remain an issue in DNA metabarcoding due to chimera formation). We conclude that the common practice of increasing the number of PCR cycles should not negatively impact the outcome of a high-throughput DNA barcoding study in terms of the occurrence of point mutations.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

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Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Modeling Steroidogenesis Disruption Using High-Throughput ...

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Environmental chemicals can elicit endocrine disruption by altering steroid hormone biosynthesis and metabolism (steroidogenesis) causing adverse reproductive and developmental effects. Historically, a lack of assays resulted in few chemicals having been evaluated for effects on steroidogenesis. The steroidogenic pathway is a series of hydroxylation and dehydrogenation steps carried out by CYP450 and hydroxysteroid dehydrogenase enzymes, yet the only enzyme in the pathway for which a high-throughput screening (HTS) assay has been developed is aromatase (CYP19A1), responsible for the aromatization of androgens to estrogens. Recently, the ToxCast HTS program adapted the OECD validated H295R steroidogenesis assay using human adrenocortical carcinoma cells into a high-throughput model to quantitatively assess the concentration-dependent (0.003-100 µM) effects of chemicals on 10 steroid hormones including progestagens, androgens, estrogens and glucocorticoids. These results, in combination with two CYP19A1 inhibition assays, comprise a large dataset amenable to clustering approaches supporting the identification and characterization of putative mechanisms of action (pMOA) for steroidogenesis disruption. In total, 514 chemicals were tested in all CYP19A1 and steroidogenesis assays. 216 chemicals were identified as CYP19A1 inhibitors in at least one CYP19A1 assay. 208 of these chemicals also altered hormone levels in the H295R assay, suggesting 96% sensitivity in the

Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping

Directory of Open Access Journals (Sweden)

Prasanth VP

2006-08-01

Full Text Available Abstract Background With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. Results A laboratory information management system (LIMS has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat genotyping data from the legume (chickpea, groundnut and pigeonpea and cereal (sorghum and millets crops of importance in the semi-arid tropics. Conclusion A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping

Controlling high-throughput manufacturing at the nano-scale

Science.gov (United States)

Cooper, Khershed P.

2013-09-01

Interest in nano-scale manufacturing research and development is growing. The reason is to accelerate the translation of discoveries and inventions of nanoscience and nanotechnology into products that would benefit industry, economy and society. Ongoing research in nanomanufacturing is focused primarily on developing novel nanofabrication techniques for a variety of applications—materials, energy, electronics, photonics, biomedical, etc. Our goal is to foster the development of high-throughput methods of fabricating nano-enabled products. Large-area parallel processing and highspeed continuous processing are high-throughput means for mass production. An example of large-area processing is step-and-repeat nanoimprinting, by which nanostructures are reproduced again and again over a large area, such as a 12 in wafer. Roll-to-roll processing is an example of continuous processing, by which it is possible to print and imprint multi-level nanostructures and nanodevices on a moving flexible substrate. The big pay-off is high-volume production and low unit cost. However, the anticipated cost benefits can only be realized if the increased production rate is accompanied by high yields of high quality products. To ensure product quality, we need to design and construct manufacturing systems such that the processes can be closely monitored and controlled. One approach is to bring cyber-physical systems (CPS) concepts to nanomanufacturing. CPS involves the control of a physical system such as manufacturing through modeling, computation, communication and control. Such a closely coupled system will involve in-situ metrology and closed-loop control of the physical processes guided by physics-based models and driven by appropriate instrumentation, sensing and actuation. This paper will discuss these ideas in the context of controlling high-throughput manufacturing at the nano-scale.

Applications of High-Throughput Nucleotide Sequencing (PhD)

DEFF Research Database (Denmark)

Waage, Johannes

equally large demands in data handling, analysis and interpretation, perhaps defining the modern challenge of the computational biologist of the post-genomic era. The first part of this thesis consists of a general introduction to the history, common terms and challenges of next generation sequencing......-sequencing, a study of the effects on alternative RNA splicing of KO of the nonsense mediated RNA decay system in Mus, using digital gene expression and a custom-built exon-exon junction mapping pipeline is presented (article I). Evolved from this work, a Bioconductor package, spliceR, for classifying alternative...

Transcriptome-Wide Analysis of Botrytis elliptica Responsive microRNAs and Their Targets in Lilium Regale Wilson by High-Throughput Sequencing and Degradome Analysis

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Xue Gao

2017-05-01

Full Text Available MicroRNAs, as master regulators of gene expression, have been widely identified and play crucial roles in plant-pathogen interactions. A fatal pathogen, Botrytis elliptica, causes the serious folia disease of lily, which reduces production because of the high susceptibility of most cultivated species. However, the miRNAs related to Botrytis infection of lily, and the miRNA-mediated gene regulatory networks providing resistance to B. elliptica in lily remain largely unexplored. To systematically dissect B. elliptica-responsive miRNAs and their target genes, three small RNA libraries were constructed from the leaves of Lilium regale, a promising Chinese wild Lilium species, which had been subjected to mock B. elliptica treatment or B. elliptica infection for 6 and 24 h. By high-throughput sequencing, 71 known miRNAs belonging to 47 conserved families and 24 novel miRNA were identified, of which 18 miRNAs were downreguleted and 13 were upregulated in response to B. elliptica. Moreover, based on the lily mRNA transcriptome, 22 targets for 9 known and 1 novel miRNAs were identified by the degradome sequencing approach. Most target genes for elliptica-responsive miRNAs were involved in metabolic processes, few encoding different transcription factors, including ELONGATION FACTOR 1 ALPHA (EF1a and TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 2 (TCP2. Furthermore, the expression patterns of a set of elliptica-responsive miRNAs and their targets were validated by quantitative real-time PCR. This study represents the first transcriptome-based analysis of miRNAs responsive to B. elliptica and their targets in lily. The results reveal the possible regulatory roles of miRNAs and their targets in B. elliptica interaction, which will extend our understanding of the mechanisms of this disease in lily.

Alignment of time-resolved data from high throughput experiments.

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Abidi, Nada; Franke, Raimo; Findeisen, Peter; Klawonn, Frank

2016-12-01

To better understand the dynamics of the underlying processes in cells, it is necessary to take measurements over a time course. Modern high-throughput technologies are often used for this purpose to measure the behavior of cell products like metabolites, peptides, proteins, [Formula: see text]RNA or mRNA at different points in time. Compared to classical time series, the number of time points is usually very limited and the measurements are taken at irregular time intervals. The main reasons for this are the costs of the experiments and the fact that the dynamic behavior usually shows a strong reaction and fast changes shortly after a stimulus and then slowly converges to a certain stable state. Another reason might simply be missing values. It is common to repeat the experiments and to have replicates in order to carry out a more reliable analysis. The ideal assumptions that the initial stimulus really started exactly at the same time for all replicates and that the replicates are perfectly synchronized are seldom satisfied. Therefore, there is a need to first adjust or align the time-resolved data before further analysis is carried out. Dynamic time warping (DTW) is considered as one of the common alignment techniques for time series data with equidistant time points. In this paper, we modified the DTW algorithm so that it can align sequences with measurements at different, non-equidistant time points with large gaps in between. This type of data is usually known as time-resolved data characterized by irregular time intervals between measurements as well as non-identical time points for different replicates. This new algorithm can be easily used to align time-resolved data from high-throughput experiments and to come across existing problems such as time scarcity and existing noise in the measurements. We propose a modified method of DTW to adapt requirements imposed by time-resolved data by use of monotone cubic interpolation splines. Our presented approach

High throughput techniques to reveal the molecular physiology and evolution of digestion in spiders.

Science.gov (United States)

Fuzita, Felipe J; Pinkse, Martijn W H; Patane, José S L; Verhaert, Peter D E M; Lopes, Adriana R

2016-09-07

Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput ("-omics") techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands.

The First Report of miRNAs from a Thysanopteran Insect, Thrips palmi Karny Using High-Throughput Sequencing.

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K B Rebijith

Full Text Available Thrips palmi Karny (Thysanoptera: Thripidae is the sole vector of Watermelon bud necrosis tospovirus, where the crop loss has been estimated to be around USD 50 million annually. Chemical insecticides are of limited use in the management of T. palmi due to the thigmokinetic behaviour and development of high levels of resistance to insecticides. There is an urgent need to find out an effective futuristic management strategy, where the small RNAs especially microRNAs hold great promise as a key player in the growth and development. miRNAs are a class of short non-coding RNAs involved in regulation of gene expression either by mRNA cleavage or by translational repression. We identified and characterized a total of 77 miRNAs from T. palmi using high-throughput deep sequencing. Functional classifications of the targets for these miRNAs revealed that majority of them are involved in the regulation of transcription and translation, nucleotide binding and signal transduction. We have also validated few of these miRNAs employing stem-loop RT-PCR, qRT-PCR and Northern blot. The present study not only provides an in-depth understanding of the biological and physiological roles of miRNAs in governing gene expression but may also lead as an invaluable tool for the management of thysanopteran insects in the future.

High throughput experimentation for the discovery of new catalysts

International Nuclear Information System (INIS)

Thomson, S.; Hoffmann, C.; Johann, T.; Wolf, A.; Schmidt, H.-W.; Farrusseng, D.; Schueth, F.

2002-01-01

Full text: The use of combinatorial chemistry to obtain new materials has been developed extensively by the pharmaceutical and biochemical industries, but such approaches have been slow to impact on the field of heterogeneous catalysis. The reasons for this lie in with difficulties associated in the synthesis, characterisation and determination of catalytic properties of such materials. In many synthetic and catalytic reactions, the conditions used are difficult to emulate using High Throughput Experimentation (HTE). Furthermore, the ability to screen these catalysts simultaneously in real time, requires the development and/or modification of characterisation methods. Clearly, there is a need for both high throughput synthesis and screening of new and novel reactions, and we describe several new concepts that help to achieve these goals. Although such problems have impeded the development of combinatorial catalysis, the fact remains that many highly attractive processes still exist for which no suitable catalysts have been developed. The ability to decrease the tiFme needed to evaluate catalyst is therefore essential and this makes the use of high throughput techniques highly desirable. In this presentation we will describe the synthesis, catalytic testing, and novel screening methods developed at the Max Planck Institute. Automated synthesis procedures, performed by the use of a modified Gilson pipette robot, will be described, as will the development of two 16 and 49 sample fixed bed reactors and two 25 and 29 sample three phase reactors for catalytic testing. We will also present new techniques for the characterisation of catalysts and catalytic products using standard IR microscopy and infrared focal plane array detection, respectively

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

LENUS (Irish Health Repository)

Rawstron, A C

2016-04-01

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Identification of QTLs for 14 Agronomically Important Traits in Setaria italica Based on SNPs Generated from High-Throughput Sequencing

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Kai Zhang

2017-05-01

Full Text Available Foxtail millet (Setaria italica is an important crop possessing C4 photosynthesis capability. The S. italica genome was de novo sequenced in 2012, but the sequence lacked high-density genetic maps with agronomic and yield trait linkages. In the present study, we resequenced a foxtail millet population of 439 recombinant inbred lines (RILs and developed high-resolution bin map and high-density SNP markers, which could provide an effective approach for gene identification. A total of 59 QTL for 14 agronomic traits in plants grown under long- and short-day photoperiods were identified. The phenotypic variation explained ranged from 4.9 to 43.94%. In addition, we suggested that there may be segregation distortion on chromosome 6 that is significantly distorted toward Zhang gu. The newly identified QTL will provide a platform for sequence-based research on the S. italica genome, and for molecular marker-assisted breeding.

Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

Science.gov (United States)

Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

2017-04-01

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

High-throughput anisotropic plasma etching of polyimide for MEMS

International Nuclear Information System (INIS)

Bliznetsov, Vladimir; Manickam, Anbumalar; Ranganathan, Nagarajan; Chen, Junwei

2011-01-01

This note describes a new high-throughput process of polyimide etching for the fabrication of MEMS devices with an organic sacrificial layer approach. Using dual frequency superimposed capacitively coupled plasma we achieved a vertical profile of polyimide with an etching rate as high as 3.5 µm min −1 . After the fabrication of vertical structures in a polyimide material, additional steps were performed to fabricate structural elements of MEMS by deposition of a SiO 2 layer and performing release etching of polyimide. (technical note)

Exploring the Mechanisms of Gastrointestinal Cancer Development Using Deep Sequencing Analysis

International Nuclear Information System (INIS)

Matsumoto, Tomonori; Shimizu, Takahiro; Takai, Atsushi; Marusawa, Hiroyuki

2015-01-01

Next-generation sequencing (NGS) technologies have revolutionized cancer genomics due to their high throughput sequencing capacity. Reports of the gene mutation profiles of various cancers by many researchers, including international cancer genome research consortia, have increased over recent years. In addition to detecting somatic mutations in tumor cells, NGS technologies enable us to approach the subject of carcinogenic mechanisms from new perspectives. Deep sequencing, a method of optimizing the high throughput capacity of NGS technologies, allows for the detection of genetic aberrations in small subsets of premalignant and/or tumor cells in noncancerous chronically inflamed tissues. Genome-wide NGS data also make it possible to clarify the mutational signatures of each cancer tissue by identifying the precise pattern of nucleotide alterations in the cancer genome, providing new information regarding the mechanisms of tumorigenesis. In this review, we highlight these new methods taking advantage of NGS technologies, and discuss our current understanding of carcinogenic mechanisms elucidated from such approaches

Exploring the Mechanisms of Gastrointestinal Cancer Development Using Deep Sequencing Analysis

Energy Technology Data Exchange (ETDEWEB)

Matsumoto, Tomonori; Shimizu, Takahiro; Takai, Atsushi; Marusawa, Hiroyuki, E-mail: maru@kuhp.kyoto-u.ac.jp [Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan)

2015-06-15

Next-generation sequencing (NGS) technologies have revolutionized cancer genomics due to their high throughput sequencing capacity. Reports of the gene mutation profiles of various cancers by many researchers, including international cancer genome research consortia, have increased over recent years. In addition to detecting somatic mutations in tumor cells, NGS technologies enable us to approach the subject of carcinogenic mechanisms from new perspectives. Deep sequencing, a method of optimizing the high throughput capacity of NGS technologies, allows for the detection of genetic aberrations in small subsets of premalignant and/or tumor cells in noncancerous chronically inflamed tissues. Genome-wide NGS data also make it possible to clarify the mutational signatures of each cancer tissue by identifying the precise pattern of nucleotide alterations in the cancer genome, providing new information regarding the mechanisms of tumorigenesis. In this review, we highlight these new methods taking advantage of NGS technologies, and discuss our current understanding of carcinogenic mechanisms elucidated from such approaches.

Sequence2Vec: A novel embedding approach for modeling transcription factor binding affinity landscape

KAUST Repository

Dai, Hanjun

2017-07-26

Motivation: An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Results: Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model (HMM) which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these HMMs into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA data sets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods.

High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats.

Science.gov (United States)

Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

2015-01-27

Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. Copyright © 2015 Zhou et al.

Differential Expression and Functional Analysis of High-Throughput -Omics Data Using Open Source Tools.

Science.gov (United States)

Kebschull, Moritz; Fittler, Melanie Julia; Demmer, Ryan T; Papapanou, Panos N

2017-01-01

Today, -omics analyses, including the systematic cataloging of messenger RNA and microRNA sequences or DNA methylation patterns in a cell population, organ, or tissue sample, allow for an unbiased, comprehensive genome-level analysis of complex diseases, offering a large advantage over earlier "candidate" gene or pathway analyses. A primary goal in the analysis of these high-throughput assays is the detection of those features among several thousand that differ between different groups of samples. In the context of oral biology, our group has successfully utilized -omics technology to identify key molecules and pathways in different diagnostic entities of periodontal disease.A major issue when inferring biological information from high-throughput -omics studies is the fact that the sheer volume of high-dimensional data generated by contemporary technology is not appropriately analyzed using common statistical methods employed in the biomedical sciences.In this chapter, we outline a robust and well-accepted bioinformatics workflow for the initial analysis of -omics data generated using microarrays or next-generation sequencing technology using open-source tools. Starting with quality control measures and necessary preprocessing steps for data originating from different -omics technologies, we next outline a differential expression analysis pipeline that can be used for data from both microarray and sequencing experiments, and offers the possibility to account for random or fixed effects. Finally, we present an overview of the possibilities for a functional analysis of the obtained data.

High Throughput PBTK: Open-Source Data and Tools for ...

Science.gov (United States)

Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy

The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing

DEFF Research Database (Denmark)

Binladen, Jonas; Gilbert, M Thomas P; Bollback, Jonathan P

2007-01-01

BACKGROUND: The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine...... primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution...

High-resolution and high-throughput multichannel Fourier transform spectrometer with two-dimensional interferogram warping compensation

Science.gov (United States)

Watanabe, A.; Furukawa, H.

2018-04-01

The resolution of multichannel Fourier transform (McFT) spectroscopy is insufficient for many applications despite its extreme advantage of high throughput. We propose an improved configuration to realise both performance using a two-dimensional area sensor. For the spectral resolution, we obtained the interferogram of a larger optical path difference by shifting the area sensor without altering any optical components. The non-linear phase error of the interferometer was successfully corrected using a phase-compensation calculation. Warping compensation was also applied to realise a higher throughput to accumulate the signal between vertical pixels. Our approach significantly improved the resolution and signal-to-noise ratio by factors of 1.7 and 34, respectively. This high-resolution and high-sensitivity McFT spectrometer will be useful for detecting weak light signals such as those in non-invasive diagnosis.

Genetic Bases of Bicuspid Aortic Valve: The Contribution of Traditional and High-Throughput Sequencing Approaches on Research and Diagnosis.

Science.gov (United States)

Giusti, Betti; Sticchi, Elena; De Cario, Rosina; Magi, Alberto; Nistri, Stefano; Pepe, Guglielmina

2017-01-01

Bicuspid aortic valve (BAV) is a common (0.5-2.0% of general population) congenital heart defect with increased prevalence of aortic dilatation and dissection. BAV has an autosomal dominant inheritance with reduced penetrance and variable expressivity. BAV has been described as an isolated trait or associated with syndromic conditions [e.g., Marfan Marfan syndrome or Loeys-Dietz syndrome (MFS, LDS)]. Identification of a syndromic condition in a BAV patient is clinically relevant to personalize aortic surgery indication. A 4-fold increase in BAV prevalence in a large cohort of unrelated MFS patients with respect to general population was reported, as well as in LDS patients (8-fold). It is also known that BAV is more frequent in patients with thoracic aortic aneurysm (TAA) related to mutations in ACTA2, FBN1 , and TGFBR2 genes. Moreover, in 8 patients with BAV and thoracic aortic dilation, not fulfilling the clinical criteria for MFS, FBN1 mutations in 2/8 patients were identified suggesting that FBN1 or other genes involved in syndromic conditions correlated to aortopathy could be involved in BAV. Beyond loci associated to syndromic disorders, studies in humans and animal models evidenced/suggested the role of further genes in non-syndromic BAV. The transcriptional regulator NOTCH1 has been associated with the development and acceleration of calcium deposition. Genome wide marker-based linkage analysis demonstrated a linkage of BAV to loci on chromosomes 18, 5, and 13q. Recently, a role for GATA4 / 5 in aortic valve morphogenesis and endocardial cell differentiation has been reported. BAV has also been associated with a reduced UFD1L gene expression or involvement of a locus containing AXIN1 / PDIA2 . Much remains to be understood about the genetics of BAV. In the last years, high-throughput sequencing technologies, allowing the analysis of large number of genes or entire exomes or genomes, progressively became available. The latter issue together with the

Genetic Bases of Bicuspid Aortic Valve: The Contribution of Traditional and High-Throughput Sequencing Approaches on Research and Diagnosis

Directory of Open Access Journals (Sweden)

Betti Giusti

2017-08-01

Full Text Available Bicuspid aortic valve (BAV is a common (0.5–2.0% of general population congenital heart defect with increased prevalence of aortic dilatation and dissection. BAV has an autosomal dominant inheritance with reduced penetrance and variable expressivity. BAV has been described as an isolated trait or associated with syndromic conditions [e.g., Marfan Marfan syndrome or Loeys-Dietz syndrome (MFS, LDS]. Identification of a syndromic condition in a BAV patient is clinically relevant to personalize aortic surgery indication. A 4-fold increase in BAV prevalence in a large cohort of unrelated MFS patients with respect to general population was reported, as well as in LDS patients (8-fold. It is also known that BAV is more frequent in patients with thoracic aortic aneurysm (TAA related to mutations in ACTA2, FBN1, and TGFBR2 genes. Moreover, in 8 patients with BAV and thoracic aortic dilation, not fulfilling the clinical criteria for MFS, FBN1 mutations in 2/8 patients were identified suggesting that FBN1 or other genes involved in syndromic conditions correlated to aortopathy could be involved in BAV. Beyond loci associated to syndromic disorders, studies in humans and animal models evidenced/suggested the role of further genes in non-syndromic BAV. The transcriptional regulator NOTCH1 has been associated with the development and acceleration of calcium deposition. Genome wide marker-based linkage analysis demonstrated a linkage of BAV to loci on chromosomes 18, 5, and 13q. Recently, a role for GATA4/5 in aortic valve morphogenesis and endocardial cell differentiation has been reported. BAV has also been associated with a reduced UFD1L gene expression or involvement of a locus containing AXIN1/PDIA2. Much remains to be understood about the genetics of BAV. In the last years, high-throughput sequencing technologies, allowing the analysis of large number of genes or entire exomes or genomes, progressively became available. The latter issue together with

High-throughput screening of tick-borne pathogens in Europe

DEFF Research Database (Denmark)

Michelet, Lorraine; Delannoy, Sabine; Devillers, Elodie

2014-01-01

was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia......, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia...

Identification of QTLs for 14 Agronomically Important Traits in Setaria italica Based on SNPs Generated from High-Throughput Sequencing.

Science.gov (United States)

Zhang, Kai; Fan, Guangyu; Zhang, Xinxin; Zhao, Fang; Wei, Wei; Du, Guohua; Feng, Xiaolei; Wang, Xiaoming; Wang, Feng; Song, Guoliang; Zou, Hongfeng; Zhang, Xiaolei; Li, Shuangdong; Ni, Xuemei; Zhang, Gengyun; Zhao, Zhihai

2017-05-05

Foxtail millet ( Setaria italica ) is an important crop possessing C4 photosynthesis capability. The S. italica genome was de novo sequenced in 2012, but the sequence lacked high-density genetic maps with agronomic and yield trait linkages. In the present study, we resequenced a foxtail millet population of 439 recombinant inbred lines (RILs) and developed high-resolution bin map and high-density SNP markers, which could provide an effective approach for gene identification. A total of 59 QTL for 14 agronomic traits in plants grown under long- and short-day photoperiods were identified. The phenotypic variation explained ranged from 4.9 to 43.94%. In addition, we suggested that there may be segregation distortion on chromosome 6 that is significantly distorted toward Zhang gu. The newly identified QTL will provide a platform for sequence-based research on the S. italica genome, and for molecular marker-assisted breeding. Copyright © 2017 Zhang et al.

Modular high-throughput test stand for versatile screening of thin-film materials libraries

International Nuclear Information System (INIS)

Thienhaus, Sigurd; Hamann, Sven; Ludwig, Alfred

2011-01-01

Versatile high-throughput characterization tools are required for the development of new materials using combinatorial techniques. Here, we describe a modular, high-throughput test stand for the screening of thin-film materials libraries, which can carry out automated electrical, magnetic and magnetoresistance measurements in the temperature range of −40 to 300 °C. As a proof of concept, we measured the temperature-dependent resistance of Fe–Pd–Mn ferromagnetic shape-memory alloy materials libraries, revealing reversible martensitic transformations and the associated transformation temperatures. Magneto-optical screening measurements of a materials library identify ferromagnetic samples, whereas resistivity maps support the discovery of new phases. A distance sensor in the same setup allows stress measurements in materials libraries deposited on cantilever arrays. A combination of these methods offers a fast and reliable high-throughput characterization technology for searching for new materials. Using this approach, a composition region has been identified in the Fe–Pd–Mn system that combines ferromagnetism and martensitic transformation.

Statistical Methods for Comparative Phenomics Using High-Throughput Phenotype Microarrays

KAUST Repository

Sturino, Joseph

2010-01-24

We propose statistical methods for comparing phenomics data generated by the Biolog Phenotype Microarray (PM) platform for high-throughput phenotyping. Instead of the routinely used visual inspection of data with no sound inferential basis, we develop two approaches. The first approach is based on quantifying the distance between mean or median curves from two treatments and then applying a permutation test; we also consider a permutation test applied to areas under mean curves. The second approach employs functional principal component analysis. Properties of the proposed methods are investigated on both simulated data and data sets from the PM platform.

Discriminating activated sludge flocs from biofilm microbial communities in a novel pilot-scale reciprocation MBR using high-throughput 16S rRNA gene sequencing.

Science.gov (United States)

De Sotto, Ryan; Ho, Jaeho; Lee, Woonyoung; Bae, Sungwoo

2018-03-29

Membrane bioreactors (MBRs) are a well-established filtration technology that has become a popular solution for treating wastewater. One of the drawbacks of MBRs, however, is the formation of biofilm on the surface of membrane modules. The occurrence of biofilms leads to biofouling, which eventually compromises water quality and damages the membranes. To prevent this, it is vital to understand the mechanism of biofilm formation on membrane surfaces. In this pilot-scale study, a novel reciprocation membrane bioreactor was operated for a period of 8 months and fed with domestic wastewater from an aerobic tank of a local WWTP. Water quality parameters were monitored and the microbial composition of the attached biofilm and suspended aggregates was evaluated in this reciprocating MBR configuration. The abundance of nitrifiers and composition of microbial communities from biofilm and suspended solids samples were investigated using qPCR and high throughput 16S amplicon sequencing. Removal efficiencies of 29%, 16%, and 15% of chemical oxygen demand, total phosphorus and total nitrogen from the influent were observed after the MBR process with average effluent concentrations of 16 mg/L, 4.6 mg/L, and 5.8 mg/L respectively. This suggests that the energy-efficient MBR, apart from reducing the total energy consumption, was able to maintain effluent concentrations that are within regulatory standards for discharge. Molecular analysis showed the presence of amoA Bacteria and 16S Nitrospira genes with the occurrence of nitrification. Candidatus Accumulibacter, a genus with organisms that can accumulate phosphorus, was found to be present in both groups which explains why phosphorus removal was observed in the system. High-throughput 16S rRNA amplicon sequencing revealed the genus Saprospira to be the most abundant species from the total OTUs of both the membrane tank and biofilm samples. Copyright © 2018 Elsevier Ltd. All rights reserved.

Southern-by-Sequencing: A Robust Screening Approach for Molecular Characterization of Genetically Modified Crops

Directory of Open Access Journals (Sweden)

Gina M. Zastrow-Hayes

2015-03-01

Full Text Available Molecular characterization of events is an integral part of the advancement process during genetically modified (GM crop product development. Assessment of these events is traditionally accomplished by polymerase chain reaction (PCR and Southern blot analyses. Southern blot analysis can be time-consuming and comparatively expensive and does not provide sequence-level detail. We have developed a sequence-based application, Southern-by-Sequencing (SbS, utilizing sequence capture coupled with next-generation sequencing (NGS technology to replace Southern blot analysis for event selection in a high-throughput molecular characterization environment. SbS is accomplished by hybridizing indexed and pooled whole-genome DNA libraries from GM plants to biotinylated probes designed to target the sequence of transformation plasmids used to generate events within the pool. This sequence capture process enriches the sequence data obtained for targeted regions of interest (transformation plasmid DNA. Taking advantage of the DNA adjacent to the targeted bases (referred to as next-to-target sequence that accompanies the targeted transformation plasmid sequence, the data analysis detects plasmid-to-genome and plasmid-to-plasmid junctions introduced during insertion into the plant genome. Analysis of these junction sequences provides sequence-level information as to the following: the number of insertion loci including detection of unlinked, independently segregating, small DNA fragments; copy number; rearrangements, truncations, or deletions of the intended insertion DNA; and the presence of transformation plasmid backbone sequences. This molecular evidence from SbS analysis is used to characterize and select GM plants meeting optimal molecular characterization criteria. SbS technology has proven to be a robust event screening tool for use in a high-throughput molecular characterization environment.

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach.

Science.gov (United States)

Brown, Bonnie L; Watson, Mick; Minot, Samuel S; Rivera, Maria C; Franklin, Rima B

2017-03-01

to 98% assignment accuracy at the species level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at 99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities. © The

Distribution and Diversity of Bacteria and Fungi Colonization in Stone Monuments Analyzed by High-Throughput Sequencing.

Science.gov (United States)

Li, Qiang; Zhang, Bingjian; He, Zhang; Yang, Xiaoru

The historical and cultural heritage of Qingxing palace and Lingyin and Kaihua temple, located in Hangzhou of China, include a large number of exquisite Buddhist statues and ancient stone sculptures which date back to the Northern Song (960-1219 A.D.) and Qing dynasties (1636-1912 A.D.) and are considered to be some of the best examples of ancient stone sculpting techniques. They were added to the World Heritage List in 2011 because of their unique craftsmanship and importance to the study of ancient Chinese Buddhist culture. However, biodeterioration of the surface of the ancient Buddhist statues and white marble pillars not only severely impairs their aesthetic value but also alters their material structure and thermo-hygric properties. In this study, high-throughput sequencing was utilized to identify the microbial communities colonizing the stone monuments. The diversity and distribution of the microbial communities in six samples collected from three different environmental conditions with signs of deterioration were analyzed by means of bioinformatics software and diversity indices. In addition, the impact of environmental factors, including temperature, light intensity, air humidity, and the concentration of NO2 and SO2, on the microbial communities' diversity and distribution was evaluated. The results indicate that the presence of predominantly phototrophic microorganisms was correlated with light and humidity, while nitrifying bacteria and Thiobacillus were associated with NO2 and SO2 from air pollution.

High throughput screening method for assessing heterogeneity of microorganisms

NARCIS (Netherlands)

Ingham, C.J.; Sprenkels, A.J.; van Hylckama Vlieg, J.E.T.; Bomer, Johan G.; de Vos, W.M.; van den Berg, Albert

2006-01-01

The invention relates to the field of microbiology. Provided is a method which is particularly powerful for High Throughput Screening (HTS) purposes. More specific a high throughput method for determining heterogeneity or interactions of microorganisms is provided.

Adaptation to high throughput batch chromatography enhances multivariate screening.

Science.gov (United States)

Barker, Gregory A; Calzada, Joseph; Herzer, Sibylle; Rieble, Siegfried

2015-09-01

High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-Throughput Analysis With 96-Capillary Array Electrophoresis and Integrated Sample Preparation for DNA Sequencing Based on Laser Induced Fluorescence Detection

Energy Technology Data Exchange (ETDEWEB)

Xue, Gang [Iowa State Univ., Ames, IA (United States)

2001-01-01

The purpose of this research was to improve the fluorescence detection for the multiplexed capillary array electrophoresis, extend its use beyond the genomic analysis, and to develop an integrated micro-sample preparation system for high-throughput DNA sequencing. The authors first demonstrated multiplexed capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations in a 96-capillary array system with laser-induced fluorescence detection. Migration times of four kinds of fluoresceins and six polyaromatic hydrocarbons (PAHs) are normalized to one of the capillaries using two internal standards. The relative standard deviations (RSD) after normalization are 0.6-1.4% for the fluoresceins and 0.1-1.5% for the PAHs. Quantitative calibration of the separations based on peak areas is also performed, again with substantial improvement over the raw data. This opens up the possibility of performing massively parallel separations for high-throughput chemical analysis for process monitoring, combinatorial synthesis, and clinical diagnosis. The authors further improved the fluorescence detection by step laser scanning. A computer-controlled galvanometer scanner is adapted for scanning a focused laser beam across a 96-capillary array for laser-induced fluorescence detection. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries. The limit of detection for fluorescein is 3 x 10^-11 M (S/N = 3) for 5-mW of total laser power scanned at 4 Hz. The observed cross-talk among capillaries is 0.2%. Advantages include the efficient utilization of light due to the high duty-cycle of step scan, good detection performance due to the reduction of stray light, ruggedness due to the small mass of the galvanometer mirror, low cost due to the simplicity of components, and flexibility due to the independent paths for excitation and emission.

Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats.

Science.gov (United States)

Armour, John A L; Palla, Raquel; Zeeuwen, Patrick L J M; den Heijer, Martin; Schalkwijk, Joost; Hollox, Edward J

2007-01-01

Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.

FRESCO: Referential compression of highly similar sequences.

Science.gov (United States)

Wandelt, Sebastian; Leser, Ulf

2013-01-01

In many applications, sets of similar texts or sequences are of high importance. Prominent examples are revision histories of documents or genomic sequences. Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever-increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. In this paper, we propose a general open-source framework to compress large amounts of biological sequence data called Framework for REferential Sequence COmpression (FRESCO). Our basic compression algorithm is shown to be one to two orders of magnitudes faster than comparable related work, while achieving similar compression ratios. We also propose several techniques to further increase compression ratios, while still retaining the advantage in speed: 1) selecting a good reference sequence; and 2) rewriting a reference sequence to allow for better compression. In addition,we propose a new way of further boosting the compression ratios by applying referential compression to already referentially compressed files (second-order compression). This technique allows for compression ratios way beyond state of the art, for instance,4,000:1 and higher for human genomes. We evaluate our algorithms on a large data set from three different species (more than 1,000 genomes, more than 3 TB) and on a collection of versions of Wikipedia pages. Our results show that real-time compression of highly similar sequences at high compression ratios is possible on modern hardware.

Taxonomy of anaerobic digestion microbiome reveals biases associated with the applied high throughput sequencing strategies

DEFF Research Database (Denmark)

Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis

2018-01-01

In the past few years, many studies investigated the anaerobic digestion microbiome by means of 16S rRNA amplicon sequencing. Results obtained from these studies were compared to each other without taking into consideration the followed procedure for amplicons preparation and data analysis...... specifically, the microbial compositions of three laboratory scale biogas reactors were analyzed before and after addition of sodium oleate by sequencing the microbiome with three different approaches: 16S rRNA amplicon sequencing, shotgun DNA and shotgun RNA. This comparative analysis revealed that......, in amplicon sequencing, abundance of some taxa (Euryarchaeota and Spirochaetes) was biased by the inefficiency of universal primers to hybridize all the templates. Reliability of the results obtained was also influenced by the number of hypervariable regions under investigation. Finally, amplicon sequencing...

Efficient DNA fingerprinting based on the targeted sequencing of active retrotransposon insertion sites using a bench-top high-throughput sequencing platform.

Science.gov (United States)

Monden, Yuki; Yamamoto, Ayaka; Shindo, Akiko; Tahara, Makoto

2014-10-01

In many crop species, DNA fingerprinting is required for the precise identification of cultivars to protect the rights of breeders. Many families of retrotransposons have multiple copies throughout the eukaryotic genome and their integrated copies are inherited genetically. Thus, their insertion polymorphisms among cultivars are useful for DNA fingerprinting. In this study, we conducted a DNA fingerprinting based on the insertion polymorphisms of active retrotransposon families (Rtsp-1 and LIb) in sweet potato. Using 38 cultivars, we identified 2,024 insertion sites in the two families with an Illumina MiSeq sequencing platform. Of these insertion sites, 91.4% appeared to be polymorphic among the cultivars and 376 cultivar-specific insertion sites were identified, which were converted directly into cultivar-specific sequence-characterized amplified region (SCAR) markers. A phylogenetic tree was constructed using these insertion sites, which corresponded well with known pedigree information, thereby indicating their suitability for genetic diversity studies. Thus, the genome-wide comparative analysis of active retrotransposon insertion sites using the bench-top MiSeq sequencing platform is highly effective for DNA fingerprinting without any requirement for whole genome sequence information. This approach may facilitate the development of practical polymerase chain reaction-based cultivar diagnostic system and could also be applied to the determination of genetic relationships. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

OpenAIRE

Salvo-Chirnside, Eliane; Kane, Steven; Kerr, Lorraine E

2011-01-01

Abstract The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated p...

High throughput "omics" approaches to assess the effects of phytochemicals in human health studies

Czech Academy of Sciences Publication Activity Database

Ovesná, J.; Slabý, O.; Toussaint, O.; Kodíček, M.; Maršík, Petr; Pouchová, V.; Vaněk, Tomáš

2008-01-01

Roč. 99, E-S1 (2008), ES127-ES134 ISSN 0007-1145 R&D Projects: GA MŠk(CZ) 1P05OC054 Institutional research plan: CEZ:AV0Z50380511 Keywords : Nutrigenomics * Phytochemicals * High throughput platforms Subject RIV: GM - Food Processing Impact factor: 2.764, year: 2008

Use of Threshold of Toxicological Concern (TTC) with High Throughput Exposure Predictions as a Risk-Based Screening Approach to Prioritize More Than Seven Thousand Chemicals (ASCCT)

Science.gov (United States)

Here, we present results of an approach for risk-based prioritization using the Threshold of Toxicological Concern (TTC) combined with high-throughput exposure (HTE) modelling. We started with 7968 chemicals with calculated population median oral daily intakes characterized by an...

High-throughput sample adaptive offset hardware architecture for high-efficiency video coding

Science.gov (United States)

Zhou, Wei; Yan, Chang; Zhang, Jingzhi; Zhou, Xin

2018-03-01

A high-throughput hardware architecture for a sample adaptive offset (SAO) filter in the high-efficiency video coding video coding standard is presented. First, an implementation-friendly and simplified bitrate estimation method of rate-distortion cost calculation is proposed to reduce the computational complexity in the mode decision of SAO. Then, a high-throughput VLSI architecture for SAO is presented based on the proposed bitrate estimation method. Furthermore, multiparallel VLSI architecture for in-loop filters, which integrates both deblocking filter and SAO filter, is proposed. Six parallel strategies are applied in the proposed in-loop filters architecture to improve the system throughput and filtering speed. Experimental results show that the proposed in-loop filters architecture can achieve up to 48% higher throughput in comparison with prior work. The proposed architecture can reach a high-operating clock frequency of 297 MHz with TSMC 65-nm library and meet the real-time requirement of the in-loop filters for 8 K × 4 K video format at 132 fps.

Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

Science.gov (United States)

Wang, Xixian; Ren, Lihui; Su, Yetian; Ji, Yuetong; Liu, Yaoping; Li, Chunyu; Li, Xunrong; Zhang, Yi; Wang, Wei; Hu, Qiang; Han, Danxiang; Xu, Jian; Ma, Bo

2017-11-21

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

Genome-wide SNP identification by high-throughput sequencing and selective mapping allows sequence assembly positioning using a framework genetic linkage map

Directory of Open Access Journals (Sweden)

Xu Xiangming

2010-12-01

Full Text Available Abstract Background Determining the position and order of contigs and scaffolds from a genome assembly within an organism's genome remains a technical challenge in a majority of sequencing projects. In order to exploit contemporary technologies for DNA sequencing, we developed a strategy for whole genome single nucleotide polymorphism sequencing allowing the positioning of sequence contigs onto a linkage map using the bin mapping method. Results The strategy was tested on a draft genome of the fungal pathogen Venturia inaequalis, the causal agent of apple scab, and further validated using sequence contigs derived from the diploid plant genome Fragaria vesca. Using our novel method we were able to anchor 70% and 92% of sequences assemblies for V. inaequalis and F. vesca, respectively, to genetic linkage maps. Conclusions We demonstrated the utility of this approach by accurately determining the bin map positions of the majority of the large sequence contigs from each genome sequence and validated our method by mapping single sequence repeat markers derived from sequence contigs on a full mapping population.

Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

Science.gov (United States)

How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

2015-07-01

Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

CoLIde: a bioinformatics tool for CO-expression-based small RNA Loci Identification using high-throughput sequencing data.

Science.gov (United States)

Mohorianu, Irina; Stocks, Matthew Benedict; Wood, John; Dalmay, Tamas; Moulton, Vincent

2013-07-01

Small RNAs (sRNAs) are 20-25 nt non-coding RNAs that act as guides for the highly sequence-specific regulatory mechanism known as RNA silencing. Due to the recent increase in sequencing depth, a highly complex and diverse population of sRNAs in both plants and animals has been revealed. However, the exponential increase in sequencing data has also made the identification of individual sRNA transcripts corresponding to biological units (sRNA loci) more challenging when based exclusively on the genomic location of the constituent sRNAs, hindering existing approaches to identify sRNA loci. To infer the location of significant biological units, we propose an approach for sRNA loci detection called CoLIde (Co-expression based sRNA Loci Identification) that combines genomic location with the analysis of other information such as variation in expression levels (expression pattern) and size class distribution. For CoLIde, we define a locus as a union of regions sharing the same pattern and located in close proximity on the genome. Biological relevance, detected through the analysis of size class distribution, is also calculated for each locus. CoLIde can be applied on ordered (e.g., time-dependent) or un-ordered (e.g., organ, mutant) series of samples both with or without biological/technical replicates. The method reliably identifies known types of loci and shows improved performance on sequencing data from both plants (e.g., A. thaliana, S. lycopersicum) and animals (e.g., D. melanogaster) when compared with existing locus detection techniques. CoLIde is available for use within the UEA Small RNA Workbench which can be downloaded from: http://srna-workbench.cmp.uea.ac.uk.

Exploring the sources of bacterial spoilers in beefsteaks by culture-independent high-throughput sequencing.

Directory of Open Access Journals (Sweden)

Francesca De Filippis

Full Text Available Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01 indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association

Development of scalable high throughput fermentation approaches for physiological characterisation of yeast and filamentous fungi

DEFF Research Database (Denmark)

Knudsen, Peter Boldsen

producing the heterologous model polyketide, 6-methylsalicylic acid (6-MSA). An automated methodology for high throughput screening focusing on growth rates, together with a fully automated method for quantitative physiological characterisation in microtiter plates, was established for yeast. Full...

High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

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Gomes Paula

2010-10-01

Full Text Available Abstract Background Bathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology. Results A normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR and their RNA transcription level by quantitative PCR (q

Machine learning in computational biology to accelerate high-throughput protein expression.

Science.gov (United States)

Sastry, Anand; Monk, Jonathan; Tegel, Hanna; Uhlen, Mathias; Palsson, Bernhard O; Rockberg, Johan; Brunk, Elizabeth

2017-08-15

The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template for analysis of further expression and solubility datasets. ebrunk@ucsd.edu or johanr@biotech.kth.se. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Comparative high-throughput transcriptome sequencing and development of SiESTa, the Silene EST annotation database

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Marais Gabriel AB

2011-07-01

Full Text Available Abstract Background The genus Silene is widely used as a model system for addressing ecological and evolutionary questions in plants, but advances in using the genus as a model system are impeded by the lack of available resources for studying its genome. Massively parallel sequencing cDNA has recently developed into an efficient method for characterizing the transcriptomes of non-model organisms, generating massive amounts of data that enable the study of multiple species in a comparative framework. The sequences generated provide an excellent resource for identifying expressed genes, characterizing functional variation and developing molecular markers, thereby laying the foundations for future studies on gene sequence and gene expression divergence. Here, we report the results of a comparative transcriptome sequencing study of eight individuals representing four Silene and one Dianthus species as outgroup. All sequences and annotations have been deposited in a newly developed and publicly available database called SiESTa, the Silene EST annotation database. Results A total of 1,041,122 EST reads were generated in two runs on a Roche GS-FLX 454 pyrosequencing platform. EST reads were analyzed separately for all eight individuals sequenced and were assembled into contigs using TGICL. These were annotated with results from BLASTX searches and Gene Ontology (GO terms, and thousands of single-nucleotide polymorphisms (SNPs were characterized. Unassembled reads were kept as singletons and together with the contigs contributed to the unigenes characterized in each individual. The high quality of unigenes is evidenced by the proportion (49% that have significant hits in similarity searches with the A. thaliana proteome. The SiESTa database is accessible at http://www.siesta.ethz.ch. Conclusion The sequence collections established in the present study provide an important genomic resource for four Silene and one Dianthus species and will help to

Comparative high-throughput transcriptome sequencing and development of SiESTa, the Silene EST annotation database

Science.gov (United States)

2011-01-01

Background The genus Silene is widely used as a model system for addressing ecological and evolutionary questions in plants, but advances in using the genus as a model system are impeded by the lack of available resources for studying its genome. Massively parallel sequencing cDNA has recently developed into an efficient method for characterizing the transcriptomes of non-model organisms, generating massive amounts of data that enable the study of multiple species in a comparative framework. The sequences generated provide an excellent resource for identifying expressed genes, characterizing functional variation and developing molecular markers, thereby laying the foundations for future studies on gene sequence and gene expression divergence. Here, we report the results of a comparative transcriptome sequencing study of eight individuals representing four Silene and one Dianthus species as outgroup. All sequences and annotations have been deposited in a newly developed and publicly available database called SiESTa, the Silene EST annotation database. Results A total of 1,041,122 EST reads were generated in two runs on a Roche GS-FLX 454 pyrosequencing platform. EST reads were analyzed separately for all eight individuals sequenced and were assembled into contigs using TGICL. These were annotated with results from BLASTX searches and Gene Ontology (GO) terms, and thousands of single-nucleotide polymorphisms (SNPs) were characterized. Unassembled reads were kept as singletons and together with the contigs contributed to the unigenes characterized in each individual. The high quality of unigenes is evidenced by the proportion (49%) that have significant hits in similarity searches with the A. thaliana proteome. The SiESTa database is accessible at http://www.siesta.ethz.ch. Conclusion The sequence collections established in the present study provide an important genomic resource for four Silene and one Dianthus species and will help to further develop Silene as a

Applications of ambient mass spectrometry in high-throughput screening.

Science.gov (United States)

Li, Li-Ping; Feng, Bao-Sheng; Yang, Jian-Wang; Chang, Cui-Lan; Bai, Yu; Liu, Hu-Wei

2013-06-07

The development of rapid screening and identification techniques is of great importance for drug discovery, doping control, forensic identification, food safety and quality control. Ambient mass spectrometry (AMS) allows rapid and direct analysis of various samples in open air with little sample preparation. Recently, its applications in high-throughput screening have been in rapid progress. During the past decade, various ambient ionization techniques have been developed and applied in high-throughput screening. This review discusses typical applications of AMS, including DESI (desorption electrospray ionization), DART (direct analysis in real time), EESI (extractive electrospray ionization), etc., in high-throughput screening (HTS).

Characterization of Bacterial and Fungal Community Dynamics by High-Throughput Sequencing (HTS Metabarcoding during Flax Dew-Retting

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Christophe Djemiel

2017-10-01

Full Text Available Flax dew-retting is a key step in the industrial extraction of fibers from flax stems and is dependent upon the production of a battery of hydrolytic enzymes produced by micro-organisms during this process. To explore the diversity and dynamics of bacterial and fungal communities involved in this process we applied a high-throughput sequencing (HTS DNA metabarcoding approach (16S rRNA/ITS region, Illumina Miseq on plant and soil samples obtained over a period of 7 weeks in July and August 2014. Twenty-three bacterial and six fungal phyla were identified in soil samples and 11 bacterial and four fungal phyla in plant samples. Dominant phyla were Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes (bacteria and Ascomycota, Basidiomycota, and Zygomycota (fungi all of which have been previously associated with flax dew-retting except for Bacteroidetes and Basidiomycota that were identified for the first time. Rare phyla also identified for the first time in this process included Acidobacteria, CKC4, Chlorobi, Fibrobacteres, Gemmatimonadetes, Nitrospirae and TM6 (bacteria, and Chytridiomycota (fungi. No differences in microbial communities and colonization dynamics were observed between early and standard flax harvests. In contrast, the common agricultural practice of swath turning affects both bacterial and fungal community membership and structure in straw samples and may contribute to a more uniform retting. Prediction of community function using PICRUSt indicated the presence of a large collection of potential bacterial enzymes capable of hydrolyzing backbones and side-chains of cell wall polysaccharides. Assignment of functional guild (functional group using FUNGuild software highlighted a change from parasitic to saprophytic trophic modes in fungi during retting. This work provides the first exhaustive description of the microbial communities involved in flax dew-retting and will provide a valuable benchmark in future studies aiming

High-throughput microfluidics automated cytogenetic processing for effectively lowering biological process time and aid triage during radiation accidents

International Nuclear Information System (INIS)

Ramakumar, Adarsh

2016-01-01

Nuclear or radiation mass casualties require individual, rapid, and accurate dose-based triage of exposed subjects for cytokine therapy and supportive care, to save life. Radiation mass casualties will demand high-throughput individual diagnostic dose assessment for medical management of exposed subjects. Cytogenetic techniques are widely used for triage and definitive radiation biodosimetry. Prototype platform to demonstrate high-throughput microfluidic micro incubation to support the logistics of sample in miniaturized incubators from the site of accident to analytical labs has been developed. Efforts have been made, both at the level of developing concepts and advanced system for higher throughput in processing the samples and also implementing better and efficient methods of logistics leading to performance of lab-on-chip analyses. Automated high-throughput platform with automated feature extraction, storage, cross platform data linkage, cross platform validation and inclusion of multi-parametric biomarker approaches will provide the first generation high-throughput platform systems for effective medical management, particularly during radiation mass casualty events

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

KAUST Repository

Shokry, Ahmed M.

2014-02-01

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

Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

Science.gov (United States)

Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

2013-07-01

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

Characterization of Intestinal Microbiomes of Hirschsprung's Disease Patients with or without Enterocolitis Using Illumina-MiSeq High-Throughput Sequencing.

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Yuqing Li

Full Text Available Hirschsprung-associated enterocolitis (HAEC is a life-threatening complication of Hirschsprung's disease (HD. Although the pathological mechanisms are still unclear, studies have shown that HAEC has a close relationship with the disturbance of intestinal microbiota. This study aimed to investigate the characteristics of the intestinal microbiome of HD patients with or without enterocolitis. During routine or emergency surgery, we collected 35 intestinal content samples from five patients with HAEC and eight HD patients, including three HD patients with a history of enterocolitis who were in a HAEC remission (HAEC-R phase. Using Illumina-MiSeq high-throughput sequencing, we sequenced the V4 region of bacterial 16S rRNA, and operational taxonomic units (OTUs were defined by 97% sequence similarity. Principal coordinate analysis (PCoA of weighted UniFrac distances was performed to evaluate the diversity of each intestinal microbiome sample. The microbiota differed significantly between the HD patients (characterized by the prevalence of Bacteroidetes and HAEC patients (characterized by the prevalence of Proteobacteria, while the microbiota of the HAEC-R patients was more similar to that of the HAEC patients. We also observed that the specimens from different intestinal sites of each HD patient differed significantly, while the specimens from different intestinal sites of each HAEC and HAEC-R patient were more similar. In conclusion, the microbiome pattern of the HAEC-R patients was more similar to that of the HAEC patients than to that of the HD patients. The HD patients had a relatively distinct, more stable community than the HAEC and HAEC-R patients, suggesting that enterocolitis may either be caused by or result in a disruption of the patient's uniquely adapted intestinal flora. The intestinal microbiota associated with enterocolitis may persist following symptom resolution and can be implicated in the symptom recurrence.

High-throughput screening (HTS) and modeling of the retinoid ...

Science.gov (United States)

Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system Presentation at the Retinoids Review 2nd workshop in Brussels, Belgium on the application of high throughput screening and model to the retinoid system

Distribution and Diversity of Bacteria and Fungi Colonization in Stone Monuments Analyzed by High-Throughput Sequencing.

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Qiang Li

Full Text Available The historical and cultural heritage of Qingxing palace and Lingyin and Kaihua temple, located in Hangzhou of China, include a large number of exquisite Buddhist statues and ancient stone sculptures which date back to the Northern Song (960-1219 A.D. and Qing dynasties (1636-1912 A.D. and are considered to be some of the best examples of ancient stone sculpting techniques. They were added to the World Heritage List in 2011 because of their unique craftsmanship and importance to the study of ancient Chinese Buddhist culture. However, biodeterioration of the surface of the ancient Buddhist statues and white marble pillars not only severely impairs their aesthetic value but also alters their material structure and thermo-hygric properties. In this study, high-throughput sequencing was utilized to identify the microbial communities colonizing the stone monuments. The diversity and distribution of the microbial communities in six samples collected from three different environmental conditions with signs of deterioration were analyzed by means of bioinformatics software and diversity indices. In addition, the impact of environmental factors, including temperature, light intensity, air humidity, and the concentration of NO2 and SO2, on the microbial communities' diversity and distribution was evaluated. The results indicate that the presence of predominantly phototrophic microorganisms was correlated with light and humidity, while nitrifying bacteria and Thiobacillus were associated with NO2 and SO2 from air pollution.

Application of whole genome shotgun sequencing for detection and characterization of genetically modified organisms and derived products.

Science.gov (United States)

Holst-Jensen, Arne; Spilsberg, Bjørn; Arulandhu, Alfred J; Kok, Esther; Shi, Jianxin; Zel, Jana

2016-07-01

The emergence of high-throughput, massive or next-generation sequencing technologies has created a completely new foundation for molecular analyses. Various selective enrichment processes are commonly applied to facilitate detection of predefined (known) targets. Such approaches, however, inevitably introduce a bias and are prone to miss unknown targets. Here we review the application of high-throughput sequencing technologies and the preparation of fit-for-purpose whole genome shotgun sequencing libraries for the detection and characterization of genetically modified and derived products. The potential impact of these new sequencing technologies for the characterization, breeding selection, risk assessment, and traceability of genetically modified organisms and genetically modified products is yet to be fully acknowledged. The published literature is reviewed, and the prospects for future developments and use of the new sequencing technologies for these purposes are discussed.

A New Statistical Approach to Characterize Chemical-Elicited Behavioral Effects in High-Throughput Studies Using Zebrafish.

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Guozhu Zhang

Full Text Available Zebrafish have become an important alternative model for characterizing chemical bioactivity, partly due to the efficiency at which systematic, high-dimensional data can be generated. However, these new data present analytical challenges associated with scale and diversity. We developed a novel, robust statistical approach to characterize chemical-elicited effects in behavioral data from high-throughput screening (HTS of all 1,060 Toxicity Forecaster (ToxCast™ chemicals across 5 concentrations at 120 hours post-fertilization (hpf. Taking advantage of the immense scale of data for a global view, we show that this new approach reduces bias introduced by extreme values yet allows for diverse response patterns that confound the application of traditional statistics. We have also shown that, as a summary measure of response for local tests of chemical-associated behavioral effects, it achieves a significant reduction in coefficient of variation compared to many traditional statistical modeling methods. This effective increase in signal-to-noise ratio augments statistical power and is observed across experimental periods (light/dark conditions that display varied distributional response patterns. Finally, we integrated results with data from concomitant developmental endpoint measurements to show that appropriate statistical handling of HTS behavioral data can add important biological context that informs mechanistic hypotheses.

A high-throughput protocol for mutation scanning of the BRCA1 and BRCA2 genes

International Nuclear Information System (INIS)

Hondow, Heather L; Fox, Stephen B; Mitchell, Gillian; Scott, Rodney J; Beshay, Victoria; Wong, Stephen Q; Dobrovic, Alexander

2011-01-01

Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput. 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays. This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved

Single-nucleotide polymorphism discovery by high-throughput sequencing in sorghum

Directory of Open Access Journals (Sweden)

White Frank F

2011-07-01

Full Text Available Abstract Background Eight diverse sorghum (Sorghum bicolor L. Moench accessions were subjected to short-read genome sequencing to characterize the distribution of single-nucleotide polymorphisms (SNPs. Two strategies were used for DNA library preparation. Missing SNP genotype data were imputed by local haplotype comparison. The effect of library type and genomic diversity on SNP discovery and imputation are evaluated. Results Alignment of eight genome equivalents (6 Gb to the public reference genome revealed 283,000 SNPs at ≥82% confirmation probability. Sequencing from libraries constructed to limit sequencing to start at defined restriction sites led to genotyping 10-fold more SNPs in all 8 accessions, and correctly imputing 11% more missing data, than from semirandom libraries. The SNP yield advantage of the reduced-representation method was less than expected, since up to one fifth of reads started at noncanonical restriction sites and up to one third of restriction sites predicted in silico to yield unique alignments were not sampled at near-saturation. For imputation accuracy, the availability of a genomically similar accession in the germplasm panel was more important than panel size or sequencing coverage. Conclusions A sequence quantity of 3 million 50-base reads per accession using a BsrFI library would conservatively provide satisfactory genotyping of 96,000 sorghum SNPs. For most reliable SNP-genotype imputation in shallowly sequenced genomes, germplasm panels should consist of pairs or groups of genomically similar entries. These results may help in designing strategies for economical genotyping-by-sequencing of large numbers of plant accessions.

High-throughput scoring of seed germination

NARCIS (Netherlands)

Ligterink, Wilco; Hilhorst, Henk W.M.

2017-01-01

High-throughput analysis of seed germination for phenotyping large genetic populations or mutant collections is very labor intensive and would highly benefit from an automated setup. Although very often used, the total germination percentage after a nominated period of time is not very

Identification and Analysis of Red Sea Mangrove (Avicennia marina) microRNAs by High-Throughput Sequencing and Their Association with Stress Responses

KAUST Repository

Khraiwesh, Basel; Pugalenthi, Ganesan; Fedoroff, Nina V.

2013-01-01

Although RNA silencing has been studied primarily in model plants, advances in high-throughput sequencing technologies have enabled profiling of the small RNA components of many more plant species, providing insights into the ubiquity and conservatism of some miRNA-based regulatory mechanisms. Small RNAs of 20 to 24 nucleotides (nt) are important regulators of gene transcript levels by either transcriptional or by posttranscriptional gene silencing, contributing to genome maintenance and controlling a variety of developmental and physiological processes. Here, we used deep sequencing and molecular methods to create an inventory of the small RNAs in the mangrove species, Avicennia marina. We identified 26 novel mangrove miRNAs and 193 conserved miRNAs belonging to 36 families. We determined that 2 of the novel miRNAs were produced from known miRNA precursors and 4 were likely to be species-specific by the criterion that we found no homologs in other plant species. We used qRT-PCR to analyze the expression of miRNAs and their target genes in different tissue sets and some demonstrated tissue-specific expression. Furthermore, we predicted potential targets of these putative miRNAs based on a sequence homology and experimentally validated through endonucleolytic cleavage assays. Our results suggested that expression profiles of miRNAs and their predicted targets could be useful in exploring the significance of the conservation patterns of plants, particularly in response to abiotic stress. Because of their well-developed abilities in this regard, mangroves and other extremophiles are excellent models for such exploration. © 2013 Khraiwesh et al.

Identification and analysis of red sea mangrove (Avicennia marina microRNAs by high-throughput sequencing and their association with stress responses.

Directory of Open Access Journals (Sweden)

Basel Khraiwesh

Full Text Available Although RNA silencing has been studied primarily in model plants, advances in high-throughput sequencing technologies have enabled profiling of the small RNA components of many more plant species, providing insights into the ubiquity and conservatism of some miRNA-based regulatory mechanisms. Small RNAs of 20 to 24 nucleotides (nt are important regulators of gene transcript levels by either transcriptional or by posttranscriptional gene silencing, contributing to genome maintenance and controlling a variety of developmental and physiological processes. Here, we used deep sequencing and molecular methods to create an inventory of the small RNAs in the mangrove species, Avicennia marina. We identified 26 novel mangrove miRNAs and 193 conserved miRNAs belonging to 36 families. We determined that 2 of the novel miRNAs were produced from known miRNA precursors and 4 were likely to be species-specific by the criterion that we found no homologs in other plant species. We used qRT-PCR to analyze the expression of miRNAs and their target genes in different tissue sets and some demonstrated tissue-specific expression. Furthermore, we predicted potential targets of these putative miRNAs based on a sequence homology and experimentally validated through endonucleolytic cleavage assays. Our results suggested that expression profiles of miRNAs and their predicted targets could be useful in exploring the significance of the conservation patterns of plants, particularly in response to abiotic stress. Because of their well-developed abilities in this regard, mangroves and other extremophiles are excellent models for such exploration.

Identification and Analysis of Red Sea Mangrove (Avicennia marina) microRNAs by High-Throughput Sequencing and Their Association with Stress Responses

KAUST Repository

Khraiwesh, Basel

2013-04-08

Although RNA silencing has been studied primarily in model plants, advances in high-throughput sequencing technologies have enabled profiling of the small RNA components of many more plant species, providing insights into the ubiquity and conservatism of some miRNA-based regulatory mechanisms. Small RNAs of 20 to 24 nucleotides (nt) are important regulators of gene transcript levels by either transcriptional or by posttranscriptional gene silencing, contributing to genome maintenance and controlling a variety of developmental and physiological processes. Here, we used deep sequencing and molecular methods to create an inventory of the small RNAs in the mangrove species, Avicennia marina. We identified 26 novel mangrove miRNAs and 193 conserved miRNAs belonging to 36 families. We determined that 2 of the novel miRNAs were produced from known miRNA precursors and 4 were likely to be species-specific by the criterion that we found no homologs in other plant species. We used qRT-PCR to analyze the expression of miRNAs and their target genes in different tissue sets and some demonstrated tissue-specific expression. Furthermore, we predicted potential targets of these putative miRNAs based on a sequence homology and experimentally validated through endonucleolytic cleavage assays. Our results suggested that expression profiles of miRNAs and their predicted targets could be useful in exploring the significance of the conservation patterns of plants, particularly in response to abiotic stress. Because of their well-developed abilities in this regard, mangroves and other extremophiles are excellent models for such exploration. © 2013 Khraiwesh et al.

PRIMEGENSw3: a web-based tool for high-throughput primer and probe design.

Science.gov (United States)

Kushwaha, Garima; Srivastava, Gyan Prakash; Xu, Dong

2015-01-01

Highly specific and efficient primer and probe design has been a major hurdle in many high-throughput techniques. Successful implementation of any PCR or probe hybridization technique depends on the quality of primers and probes used in terms of their specificity and cross-hybridization. Here we describe PRIMEGENSw3, a set of web-based utilities for high-throughput primer and probe design. These utilities allow users to select genomic regions and to design primer/probe for selected regions in an interactive, user-friendly, and automatic fashion. The system runs the PRIMEGENS algorithm in the back-end on the high-performance server with the stored genomic database or user-provided custom database for cross-hybridization check. Cross-hybridization is checked not only using BLAST but also by checking mismatch positions and energy calculation of potential hybridization hits. The results can be visualized online and also can be downloaded. The average success rate of primer design using PRIMEGENSw3 is ~90 %. The web server also supports primer design for methylated sequences, which is used in epigenetic studies. Stand-alone version of the software is also available for download at the website.

metaBIT, an integrative and automated metagenomic pipeline for analysing microbial profiles from high-throughput sequencing shotgun data

DEFF Research Database (Denmark)

Louvel, Guillaume; Der Sarkissian, Clio; Hanghøj, Kristian Ebbesen

2016-01-01

-throughput DNA sequencing (HTS). Here, we develop metaBIT, an open-source computational pipeline automatizing routine microbial profiling of shotgun HTS data. Customizable by the user at different stringency levels, it performs robust taxonomy-based assignment and relative abundance calculation of microbial taxa......, as well as cross-sample statistical analyses of microbial diversity distributions. We demonstrate the versatility of metaBIT within a range of published HTS data sets sampled from the environment (soil and seawater) and the human body (skin and gut), but also from archaeological specimens. We present......-friendly profiling of the microbial DNA present in HTS shotgun data sets. The applications of metaBIT are vast, from monitoring of laboratory errors and contaminations, to the reconstruction of past and present microbiota, and the detection of candidate species, including pathogens....

Viral metagenomics: Analysis of begomoviruses by illumina high-throughput sequencing

KAUST Repository

Idris, Ali; Al-Saleh, Mohammed; Piatek, Marek J.; Al-Shahwan, Ibrahim; Ali, Shahjahan; Brown, Judith K.

2014-01-01

Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant

LSGermOPA, a custom OPA of 384 EST-derived SNPs for high-throughput lettuce (Lactuca sativa L.) germplasm fingerprinting

Science.gov (United States)

We assessed the genetic diversity and population structure among 148 cultivated lettuce (Lactuca sativa L.) accessions using the high-throughput GoldenGate assay and 384 EST (Expressed Sequence Tag)-derived SNP (single nucleotide polymorphism) markers. A custom OPA (Oligo Pool All), LSGermOPA was fo...

Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

DEFF Research Database (Denmark)

Lundberg, Martin; Thorsen, Stine Buch; Assarsson, Erika

2011-01-01

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming...... sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent...

High-Throughput Tools for Characterization of Antibody Epitopes

DEFF Research Database (Denmark)

Christiansen, Anders

mapping. In Chapter 1, it was examined whether combining phage display, a traditional epitope mapping approach, with HTS would improve the method. The developed approach was successfully used to map Ara h 1 epitopes in sera from patients with peanut allergy. Notably, the sera represented difficult...... proliferation advantages. Finally, in Chapter 4, a different emerging technology, next-generation peptide microarrays, was applied for epitope mapping of major peanut allergens using sera from allergic patients. New developments in the peptide microarray have enabled a greatly increased throughput....... In this study, these improvements were utilized to characterize epitopes at high resolution, i.e. determine the importance of each residue for antibody binding, for all major peanut allergens. Epitope reactivity among patients often converged on known epitope hotspots, however the binding patterns were somewhat...

Mining environmental high-throughput sequence data sets to identify divergent amplicon clusters for phylogenetic reconstruction and morphotype visualization.

Science.gov (United States)

Gimmler, Anna; Stoeck, Thorsten

2015-08-01

Environmental high-throughput sequencing (envHTS) is a very powerful tool, which in protistan ecology is predominantly used for the exploration of diversity and its geographic and local patterns. We here used a pyrosequenced V4-SSU rDNA data set from a solar saltern pond as test case to exploit such massive protistan amplicon data sets beyond this descriptive purpose. Therefore, we combined a Swarm-based blastn network including 11 579 ciliate V4 amplicons to identify divergent amplicon clusters with targeted polymerase chain reaction (PCR) primer design for full-length small subunit of the ribosomal DNA retrieval and probe design for fluorescence in situ hybridization (FISH). This powerful strategy allows to benefit from envHTS data sets to (i) reveal the phylogenetic position of the taxon behind divergent amplicons; (ii) improve phylogenetic resolution and evolutionary history of specific taxon groups; (iii) solidly assess an amplicons (species') degree of similarity to its closest described relative; (iv) visualize the morphotype behind a divergent amplicons cluster; (v) rapidly FISH screen many environmental samples for geographic/habitat distribution and abundances of the respective organism and (vi) to monitor the success of enrichment strategies in live samples for cultivation and isolation of the respective organisms. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

3D material cytometry (3DMaC): a very high-replicate, high-throughput analytical method using microfabricated, shape-specific, cell-material niches.

Science.gov (United States)

Parratt, Kirsten; Jeong, Jenny; Qiu, Peng; Roy, Krishnendu

2017-08-08

Studying cell behavior within 3D material niches is key to understanding cell biology in health and diseases, and developing biomaterials for regenerative medicine applications. Current approaches to studying these cell-material niches have low throughput and can only analyze a few replicates per experiment resulting in reduced measurement assurance and analytical power. Here, we report 3D material cytometry (3DMaC), a novel high-throughput method based on microfabricated, shape-specific 3D cell-material niches and imaging cytometry. 3DMaC achieves rapid and highly multiplexed analyses of very high replicate numbers ("n" of 10 4 -10 6 ) of 3D biomaterial constructs. 3DMaC overcomes current limitations of low "n", low-throughput, and "noisy" assays, to provide rapid and simultaneous analyses of potentially hundreds of parameters in 3D biomaterial cultures. The method is demonstrated here for a set of 85 000 events containing twelve distinct cell-biomaterial micro-niches along with robust, customized computational methods for high-throughput analytics with potentially unprecedented statistical power.

High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA.

Science.gov (United States)

De Barba, M; Miquel, C; Lobréaux, S; Quenette, P Y; Swenson, J E; Taberlet, P

2017-05-01

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies. © 2016 John Wiley & Sons Ltd.

Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science

NARCIS (Netherlands)

Breier, J.M.; Gassmann, K.; Kayser, R.; Stegeman, H.; Groot, D.de; Fritsche, E.; Shafer, T.J.

2010-01-01

In vitro, high-throughput methods have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramifications for the accuracy,

Supplementary Material for Finding the Stable Structures of N1-xWX with an Ab-initio High-Throughput Approach

Science.gov (United States)

2015-05-08

Supplementary material for “Finding the stable structures of N1−xWX with an ab - initio high-throughput approach” Michael J. Mehl∗ Center for...AND SUBTITLE Supplementary Material for ’Finding the Stable Structures of N1-xWX with an ab - initio High-throughput Approach’ 5a. CONTRACT NUMBER 5b...and J. Hafner, Ab initio molecular dynamics for open-shell transition metals, Phys. Rev. B 48, 13115–13118 (1993). 2 G. Kresse and J. Hafner, Ab initio

20180311 - High Throughput Transcriptomics: From screening to pathways (SOT 2018)

Science.gov (United States)

The EPA ToxCast effort has screened thousands of chemicals across hundreds of high-throughput in vitro screening assays. The project is now leveraging high-throughput transcriptomic (HTTr) technologies to substantially expand its coverage of biological pathways. The first HTTr sc...

High throughput label-free platform for statistical bio-molecular sensing

DEFF Research Database (Denmark)

Bosco, Filippo; Hwu, En-Te; Chen, Ching-Hsiu

2011-01-01

Sensors are crucial in many daily operations including security, environmental control, human diagnostics and patient monitoring. Screening and online monitoring require reliable and high-throughput sensing. We report on the demonstration of a high-throughput label-free sensor platform utilizing...

Sequence assembly

DEFF Research Database (Denmark)

Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

2009-01-01

Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies and...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

Theory and implementation of a very high throughput true random number generator in field programmable gate array

Energy Technology Data Exchange (ETDEWEB)

Wang, Yonggang, E-mail: wangyg@ustc.edu.cn; Hui, Cong; Liu, Chong; Xu, Chao [Department of Modern Physics, University of Science and Technology of China, Hefei 230026 (China)

2016-04-15

The contribution of this paper is proposing a new entropy extraction mechanism based on sampling phase jitter in ring oscillators to make a high throughput true random number generator in a field programmable gate array (FPGA) practical. Starting from experimental observation and analysis of the entropy source in FPGA, a multi-phase sampling method is exploited to harvest the clock jitter with a maximum entropy and fast sampling speed. This parametrized design is implemented in a Xilinx Artix-7 FPGA, where the carry chains in the FPGA are explored to realize the precise phase shifting. The generator circuit is simple and resource-saving, so that multiple generation channels can run in parallel to scale the output throughput for specific applications. The prototype integrates 64 circuit units in the FPGA to provide a total output throughput of 7.68 Gbps, which meets the requirement of current high-speed quantum key distribution systems. The randomness evaluation, as well as its robustness to ambient temperature, confirms that the new method in a purely digital fashion can provide high-speed high-quality random bit sequences for a variety of embedded applications.

Comparing the normalization methods for the differential analysis of Illumina high-throughput RNA-Seq data.

Science.gov (United States)

Li, Peipei; Piao, Yongjun; Shon, Ho Sun; Ryu, Keun Ho

2015-10-28

Recently, rapid improvements in technology and decrease in sequencing costs have made RNA-Seq a widely used technique to quantify gene expression levels. Various normalization approaches have been proposed, owing to the importance of normalization in the analysis of RNA-Seq data. A comparison of recently proposed normalization methods is required to generate suitable guidelines for the selection of the most appropriate approach for future experiments. In this paper, we compared eight non-abundance (RC, UQ, Med, TMM, DESeq, Q, RPKM, and ERPKM) and two abundance estimation normalization methods (RSEM and Sailfish). The experiments were based on real Illumina high-throughput RNA-Seq of 35- and 76-nucleotide sequences produced in the MAQC project and simulation reads. Reads were mapped with human genome obtained from UCSC Genome Browser Database. For precise evaluation, we investigated Spearman correlation between the normalization results from RNA-Seq and MAQC qRT-PCR values for 996 genes. Based on this work, we showed that out of the eight non-abundance estimation normalization methods, RC, UQ, Med, TMM, DESeq, and Q gave similar normalization results for all data sets. For RNA-Seq of a 35-nucleotide sequence, RPKM showed the highest correlation results, but for RNA-Seq of a 76-nucleotide sequence, least correlation was observed than the other methods. ERPKM did not improve results than RPKM. Between two abundance estimation normalization methods, for RNA-Seq of a 35-nucleotide sequence, higher correlation was obtained with Sailfish than that with RSEM, which was better than without using abundance estimation methods. However, for RNA-Seq of a 76-nucleotide sequence, the results achieved by RSEM were similar to without applying abundance estimation methods, and were much better than with Sailfish. Furthermore, we found that adding a poly-A tail increased alignment numbers, but did not improve normalization results. Spearman correlation analysis revealed that RC, UQ

SAMNet: a network-based approach to integrate multi-dimensional high throughput datasets.

Science.gov (United States)

Gosline, Sara J C; Spencer, Sarah J; Ursu, Oana; Fraenkel, Ernest

2012-11-01

The rapid development of high throughput biotechnologies has led to an onslaught of data describing genetic perturbations and changes in mRNA and protein levels in the cell. Because each assay provides a one-dimensional snapshot of active signaling pathways, it has become desirable to perform multiple assays (e.g. mRNA expression and phospho-proteomics) to measure a single condition. However, as experiments expand to accommodate various cellular conditions, proper analysis and interpretation of these data have become more challenging. Here we introduce a novel approach called SAMNet, for Simultaneous Analysis of Multiple Networks, that is able to interpret diverse assays over multiple perturbations. The algorithm uses a constrained optimization approach to integrate mRNA expression data with upstream genes, selecting edges in the protein-protein interaction network that best explain the changes across all perturbations. The result is a putative set of protein interactions that succinctly summarizes the results from all experiments, highlighting the network elements unique to each perturbation. We evaluated SAMNet in both yeast and human datasets. The yeast dataset measured the cellular response to seven different transition metals, and the human dataset measured cellular changes in four different lung cancer models of Epithelial-Mesenchymal Transition (EMT), a crucial process in tumor metastasis. SAMNet was able to identify canonical yeast metal-processing genes unique to each commodity in the yeast dataset, as well as human genes such as β-catenin and TCF7L2/TCF4 that are required for EMT signaling but escaped detection in the mRNA and phospho-proteomic data. Moreover, SAMNet also highlighted drugs likely to modulate EMT, identifying a series of less canonical genes known to be affected by the BCR-ABL inhibitor imatinib (Gleevec), suggesting a possible influence of this drug on EMT.

High Throughput Line-of-Sight MIMO Systems for Next Generation Backhaul Applications

Science.gov (United States)

Song, Xiaohang; Cvetkovski, Darko; Hälsig, Tim; Rave, Wolfgang; Fettweis, Gerhard; Grass, Eckhard; Lankl, Berthold

2017-09-01

The evolution to ultra-dense next generation networks requires a massive increase in throughput and deployment flexibility. Therefore, novel wireless backhaul solutions that can support these demands are needed. In this work we present an approach for a millimeter wave line-of-sight MIMO backhaul design, targeting transmission rates in the order of 100 Gbit/s. We provide theoretical foundations for the concept showcasing its potential, which are confirmed through channel measurements. Furthermore, we provide insights into the system design with respect to antenna array setup, baseband processing, synchronization, and channel equalization. Implementation in a 60 GHz demonstrator setup proves the feasibility of the system concept for high throughput backhauling in next generation networks.

XMRF: an R package to fit Markov Networks to high-throughput genetics data.

Science.gov (United States)

Wan, Ying-Wooi; Allen, Genevera I; Baker, Yulia; Yang, Eunho; Ravikumar, Pradeep; Anderson, Matthew; Liu, Zhandong

2016-08-26

Technological advances in medicine have led to a rapid proliferation of high-throughput "omics" data. Tools to mine this data and discover disrupted disease networks are needed as they hold the key to understanding complicated interactions between genes, mutations and aberrations, and epi-genetic markers. We developed an R software package, XMRF, that can be used to fit Markov Networks to various types of high-throughput genomics data. Encoding the models and estimation techniques of the recently proposed exponential family Markov Random Fields (Yang et al., 2012), our software can be used to learn genetic networks from RNA-sequencing data (counts via Poisson graphical models), mutation and copy number variation data (categorical via Ising models), and methylation data (continuous via Gaussian graphical models). XMRF is the only tool that allows network structure learning using the native distribution of the data instead of the standard Gaussian. Moreover, the parallelization feature of the implemented algorithms computes the large-scale biological networks efficiently. XMRF is available from CRAN and Github ( https://github.com/zhandong/XMRF ).

Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

DEFF Research Database (Denmark)

Urbanski, Dorian Fabian; Malolepszy, Anna; Stougaard, Jens

2012-01-01

Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis and insert......Insertion mutants facilitate functional analysis of genes, but for most plant species it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics. The main challenge is developing efficient high-throughput procedures for both mutagenesis...... plants. The identified insertions showed that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to its homogenous gene targeting and exonic insertion preference. Since LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating...... progeny generates a complete mutant population. This ease of LORE1 mutagenesis combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing, and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource....

High-Throughput Non-destructive Phenotyping of Traits that Contribute to Salinity Tolerance in Arabidopsis thaliana

KAUST Repository

Awlia, Mariam

2016-09-28

Reproducible and efficient high-throughput phenotyping approaches, combined with advances in genome sequencing, are facilitating the discovery of genes affecting plant performance. Salinity tolerance is a desirable trait that can be achieved through breeding, where most have aimed at selecting for plants that perform effective ion exclusion from the shoots. To determine overall plant performance under salt stress, it is helpful to investigate several plant traits collectively in one experimental setup. Hence, we developed a quantitative phenotyping protocol using a high-throughput phenotyping system, with RGB and chlorophyll fluorescence (ChlF) imaging, which captures the growth, morphology, color and photosynthetic performance of Arabidopsis thaliana plants in response to salt stress. We optimized our salt treatment by controlling the soil-water content prior to introducing salt stress. We investigated these traits over time in two accessions in soil at 150, 100, or 50 mM NaCl to find that the plants subjected to 100 mM NaCl showed the most prominent responses in the absence of symptoms of severe stress. In these plants, salt stress induced significant changes in rosette area and morphology, but less prominent changes in rosette coloring and photosystem II efficiency. Clustering of ChlF traits with plant growth of nine accessions maintained at 100 mM NaCl revealed that in the early stage of salt stress, salinity tolerance correlated with non-photochemical quenching processes and during the later stage, plant performance correlated with quantum yield. This integrative approach allows the simultaneous analysis of several phenotypic traits. In combination with various genetic resources, the phenotyping protocol described here is expected to increase our understanding of plant performance and stress responses, ultimately identifying genes that improve plant performance in salt stress conditions.

High-Throughput Network Communication with NetIO

CERN Document Server

Schumacher, J\\"orn; The ATLAS collaboration; Vandelli, Wainer

2016-01-01

HPC network technologies like Infiniband, TrueScale or OmniPath provide low-latency and high-throughput communication between hosts, which makes them attractive options for data-acquisition systems in large-scale high-energy physics experiments. Like HPC networks, DAQ networks are local and include a well specified number of systems. Unfortunately traditional network communication APIs for HPC clusters like MPI or PGAS target exclusively the HPC community and are not suited well for DAQ applications. It is possible to build distributed DAQ applications using low-level system APIs like Infiniband Verbs (and this has been done), but it requires a non negligible effort and expert knowledge. On the other hand, message services like 0MQ have gained popularity in the HEP community. Such APIs allow to build distributed applications with a high-level approach and provide good performance. Unfortunately their usage usually limits developers to TCP/IP-based networks. While it is possible to operate a TCP/IP stack on to...

High-Throughput and Low-Latency Network Communication with NetIO

Science.gov (United States)

Schumacher, Jörn; Plessl, Christian; Vandelli, Wainer

2017-10-01

HPC network technologies like Infiniband, TrueScale or OmniPath provide low- latency and high-throughput communication between hosts, which makes them attractive options for data-acquisition systems in large-scale high-energy physics experiments. Like HPC networks, DAQ networks are local and include a well specified number of systems. Unfortunately traditional network communication APIs for HPC clusters like MPI or PGAS exclusively target the HPC community and are not suited well for DAQ applications. It is possible to build distributed DAQ applications using low-level system APIs like Infiniband Verbs, but it requires a non-negligible effort and expert knowledge. At the same time, message services like ZeroMQ have gained popularity in the HEP community. They make it possible to build distributed applications with a high-level approach and provide good performance. Unfortunately, their usage usually limits developers to TCP/IP- based networks. While it is possible to operate a TCP/IP stack on top of Infiniband and OmniPath, this approach may not be very efficient compared to a direct use of native APIs. NetIO is a simple, novel asynchronous message service that can operate on Ethernet, Infiniband and similar network fabrics. In this paper the design and implementation of NetIO is presented and described, and its use is evaluated in comparison to other approaches. NetIO supports different high-level programming models and typical workloads of HEP applications. The ATLAS FELIX project [1] successfully uses NetIO as its central communication platform. The architecture of NetIO is described in this paper, including the user-level API and the internal data-flow design. The paper includes a performance evaluation of NetIO including throughput and latency measurements. The performance is compared against the state-of-the- art ZeroMQ message service. Performance measurements are performed in a lab environment with Ethernet and FDR Infiniband networks.

Deciphering the Diversities of Astroviruses and Noroviruses in Wastewater Treatment Plant Effluents by a High-Throughput Sequencing Method.

Science.gov (United States)

Prevost, B; Lucas, F S; Ambert-Balay, K; Pothier, P; Moulin, L; Wurtzer, S

2015-10-01

Although clinical epidemiology lists human enteric viruses to be among the primary causes of acute gastroenteritis in the human population, their circulation in the environment remains poorly investigated. These viruses are excreted by the human population into sewers and may be released into rivers through the effluents of wastewater treatment plants (WWTPs). In order to evaluate the viral diversity and loads in WWTP effluents of the Paris, France, urban area, which includes about 9 million inhabitants (approximately 15% of the French population), the seasonal occurrence of astroviruses and noroviruses in 100 WWTP effluent samples was investigated over 1 year. The coupling of these measurements with a high-throughput sequencing approach allowed the specific estimation of the diversity of human astroviruses (human astrovirus genotype 1 [HAstV-1], HAstV-2, HAstV-5, and HAstV-6), 7 genotypes of noroviruses (NoVs) of genogroup I (NoV GI.1 to NoV GI.6 and NoV GI.8), and 16 genotypes of NoVs of genogroup II (NoV GII.1 to NoV GII.7, NoV GII.9, NoV GII.12 to NoV GII.17, NoV GII.20, and NoV GII.21) in effluent samples. Comparison of the viral diversity in WWTP effluents to the viral diversity found by analysis of clinical data obtained throughout France underlined the consistency between the identified genotypes. However, some genotypes were locally present in effluents and were not found in the analysis of the clinical data. These findings could highlight an underestimation of the diversity of enteric viruses circulating in the human population. Consequently, analysis of WWTP effluents could allow the exploration of viral diversity not only in environmental waters but also in a human population linked to a sewerage network in order to better comprehend viral epidemiology and to forecast seasonal outbreaks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Peptide Pattern Recognition for high-throughput protein sequence analysis and clustering

DEFF Research Database (Denmark)

Busk, Peter Kamp

2017-01-01

Large collections of protein sequences with divergent sequences are tedious to analyze for understanding their phylogenetic or structure-function relation. Peptide Pattern Recognition is an algorithm that was developed to facilitate this task but the previous version does only allow a limited...... number of sequences as input. I implemented Peptide Pattern Recognition as a multithread software designed to handle large numbers of sequences and perform analysis in a reasonable time frame. Benchmarking showed that the new implementation of Peptide Pattern Recognition is twenty times faster than...... the previous implementation on a small protein collection with 673 MAP kinase sequences. In addition, the new implementation could analyze a large protein collection with 48,570 Glycosyl Transferase family 20 sequences without reaching its upper limit on a desktop computer. Peptide Pattern Recognition...

Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis.

Directory of Open Access Journals (Sweden)

Emmanuel Dias-Neto

2009-12-01

Full Text Available Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i the counting of transducing units and (ii the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU, with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs approximately 250-fold for generating 10(6 ligand sequences.Our analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger

Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis.