scholarly journals A novel post hoc method for detecting index switching finds no evidence for increased switching on the Illumina HiSeq X

2017 ◽  
Author(s):  
Gregory L. Owens ◽  
Marco Todesco ◽  
Emily B. M. Drummond ◽  
Sam Yeaman ◽  
Loren H. Rieseberg
Keyword(s):  
Allele Frequency ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Molecular Ecology ◽  
Whole Genome Shotgun ◽  
Whole Genome ◽  
Illumina Hiseq ◽  
Illumina Hiseq Platform ◽  
Post Hoc

AbstractHigh throughput sequencing using the Illumina HiSeq platform is a pervasive and critical molecular ecology resource, and has provided the data underlying many recent advances. A recent study has suggested that ‘index switching’, where reads are misattributed to the wrong sample, may be higher in new versions of the HiSeq platform. This has the potential to invalidate both published and in-progress work across the field. Here, we test for evidence of index switching in an exemplar whole genome shotgun dataset sequenced on both the Illumina HiSeq 2500, which should not have the problem, and the Illumina HiSeq X, which may. We leverage unbalanced heterozygotes, which may be produced by index switching, and ask whether the under-sequenced allele is more likely to be found in other samples in the same lane than expected based on the allele frequency. Although we validate the sensitivity of this method using simulations, we find that neither the HiSeq 2500 nor the HiSeq X have evidence of index switching. This suggests that, thankfully, index switching may not be a ubiquitous problem in HiSeq X sequence data. Lastly, we provide scripts for applying our method so that index switching can be tested for in other datasets.

scholarly journals Ultra-high throughput multiplexing and sequencing of >500 bp amplicon regions on the Illumina HiSeq 2500 platform

2018 ◽  
Author(s):  
Johanna B. Holm ◽  
Michael S. Humphrys ◽  
Courtney K. Robinson ◽  
Matthew L. Settles ◽  
Sandra Ott ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Microbial Community Composition ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Illumina Hiseq ◽  
Illumina Miseq Platform ◽  
Miseq Platform

AbstractAmplification, sequencing and analysis of the 16S rRNA gene affords characterization of microbial community composition. As this tool has become more popular and amplicon-sequencing applications have grown in the total number of samples, growth in sample multiplexing is becoming necessary while maintaining high sequence quality and sequencing depth. Here, modifications to the Illumina HiSeq 2500 platform are described which produce greater multiplexing capabilities and 300 bp paired-end reads of higher quality than produced by the current Illumina MiSeq platform. To improve the feasibility and flexibility of this method, a 2-Step PCR amplification protocol is also described that allows for targeting of different amplicon regions, thus improving amplification success from low bacterial bioburden samples.ImportanceAmplicon sequencing has become a popular and widespread tool for surveying microbial communities. Lower overall costs associated with high throughput sequencing have made it a widely-adopted approach, especially for projects which necessitate sample multiplexing to eliminate batch effect and reduced time to acquire data. The method for amplicon sequencing on the Illumina HiSeq 2500 platform described here provides improved multiplexing capabilities while simultaneously producing greater quality sequence data and lower per sample cost relative to the Illumina MiSeq platform, without sacrificing amplicon length. To make this method more flexible to various amplicon targeted regions as well as improve amplification from low biomass samples, we also present and validate a 2-Step PCR library preparation method.

scholarly journals Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Annika Brinkmann ◽  
Andreas Andrusch ◽  
Ariane Belka ◽  
Claudia Wylezich ◽  
Dirk Höper ◽  
...  
Keyword(s):  
Proficiency Testing ◽  
High Throughput ◽  
In Silico ◽  
High Throughput Sequencing ◽  
Bioinformatics Analysis ◽  
Sequence Data ◽  
Correct Identification ◽  
Illumina Hiseq ◽  
Data Set ◽  
Virus Diagnostics

ABSTRACT Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants’ analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

scholarly journals High-throughput sequencing of Bacillus anthracis in France: investigating genome diversity and population structure using whole-genome SNP discovery

BMC Genomics ◽  
2014 ◽  
Vol 15 (1) ◽  
pp. 288 ◽  
Author(s):  
Guillaume Girault ◽  
Yann Blouin ◽  
Gilles Vergnaud ◽  
Sylviane Derzelle
Keyword(s):  
Population Structure ◽  
Bacillus Anthracis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Genome Diversity ◽  
Snp Discovery

scholarly journals Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0254971
Author(s):  
Federico Rossi ◽  
Alessandro Crnjar ◽  
Federico Comitani ◽  
Rodrigo Feliciano ◽  
Leonie Jahn ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tree Ring ◽  
High Throughput ◽  
Environmental Conditions ◽  
High Throughput Sequencing ◽  
Wood Formation ◽  
Whole Genome ◽  
Library Preparation ◽  
Bisulfite Treatment ◽  
Standing Trees

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

scholarly journals Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems

2011 ◽  
Vol 12 (11) ◽  
pp. R112 ◽  
Author(s):  
André E Minoche ◽  
Juliane C Dohm ◽  
Heinz Himmelbauer
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Data ◽  
Illumina Hiseq ◽  
High Throughput Sequencing Data

scholarly journals Lethal Outcome of Leptospirosis in Southern Russia: Characterization of Leptospira Interrogans Isolated from a Deceased Teenager

2020 ◽  
Vol 17 (12) ◽  
pp. 4238
Author(s):  
Andrei Samoilov ◽  
Nataliya Stoyanova ◽  
Nikolai Tokarevich ◽  
Birgitta Evengard ◽  
Elena Zueva ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Agglutination Test ◽  
Leptospira Interrogans ◽  
Whole Genome ◽  
Highly Qualified ◽  
Lethal Outcome ◽  
Southern Russia

This article describes a lethal case of leptospirosis that occurred in Southern Russia. The Leptospira strain was isolated and characterized using a microscopic agglutination test, MALDI-TOF mass spectrometry, targeted PCR, and high-throughput sequencing. We show that molecular and mass-spectrometry methods can be an alternative to conventional methods of leptospirosis diagnostics and Leptospira study, which require highly qualified staff and can be performed only at specialized laboratories. We also report the first whole genome of L. interrogans isolated in Russia.

scholarly journals Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yuka Torii ◽  
Kazuhiro Horiba ◽  
Satoshi Hayano ◽  
Taichi Kato ◽  
Takako Suzuki ◽  
...  
Keyword(s):  
Kawasaki Disease ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Early Stage ◽  
Rna Virus ◽  
Systemic Vasculitis ◽  
Human Herpesvirus ◽  
Acute Stage ◽  
Serum Samples

Abstract Background Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). Methods Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. Results No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. Conclusion No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

scholarly journals High-Throughput Crystallization Pipeline at the Crystallography Core Facility of the Institut Pasteur

Molecules ◽  
2019 ◽  
Vol 24 (24) ◽  
pp. 4451 ◽  
Author(s):  
Patrick Weber ◽  
Cédric Pissis ◽  
Rafael Navaza ◽  
Ariel E. Mechaly ◽  
Frederick Saul ◽  
...  
Keyword(s):  
High Throughput ◽  
Sequence Data ◽  
Data Bank ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
X Ray Diffraction ◽  
Sequencing Technology ◽  
X Ray ◽  
Set Up ◽  
General User

The availability of whole-genome sequence data, made possible by significant advances in DNA sequencing technology, led to the emergence of structural genomics projects in the late 1990s. These projects not only significantly increased the number of 3D structures deposited in the Protein Data Bank in the last two decades, but also influenced present crystallographic strategies by introducing automation and high-throughput approaches in the structure-determination pipeline. Today, dedicated crystallization facilities, many of which are open to the general user community, routinely set up and track thousands of crystallization screening trials per day. Here, we review the current methods for high-throughput crystallization and procedures to obtain crystals suitable for X-ray diffraction studies, and we describe the crystallization pipeline implemented in the medium-scale crystallography platform at the Institut Pasteur (Paris) as an example.

Grafting-responsive miRNAs in cucumber and pumpkin seedlings identified by high-throughput sequencing at whole genome level

2013 ◽  
Vol 151 (4) ◽  
pp. 406-422 ◽  
Author(s):  
Chaohan Li ◽  
Yansu Li ◽  
Longqiang Bai ◽  
Tieyao Zhang ◽  
Chaoxing He ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Genome Level

scholarly journals Software updates in the Illumina HiSeq platform affect whole-genome bisulfite sequencing

BMC Genomics ◽  
2017 ◽  
Vol 18 (1) ◽  
Author(s):  
Hidehiro Toh ◽  
Kenjiro Shirane ◽  
Fumihito Miura ◽  
Naoki Kubo ◽  
Kenji Ichiyanagi ◽  
...  
Keyword(s):  
Bisulfite Sequencing ◽  
Whole Genome ◽  
Illumina Hiseq ◽  
Whole Genome Bisulfite Sequencing ◽  
Genome Bisulfite Sequencing ◽  
Software Updates ◽  
Illumina Hiseq Platform
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