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Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE128734 | GEO | 2019/07/29

REPOSITORIES: GEO

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Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing.

Project description:Whole genome bisulfite sequencing (WGBS), with its ability to interrogate methylation status at single CpG site resolution epigenome-wide, is a powerful technique for use in molecular experiments. Here, we aim to advance strategies for accurate and efficient WGBS for application in future large-scale epidemiological studies. We systematically compared the performance of three WGBS library preparation methods with low DNA input requirement (Swift Biosciences Accel-NGS, Illumina TruSeq and QIAGEN QIAseq) on two state-of-the-art sequencing platforms (Illumina NovaSeq and HiSeq X), and also assessed concordance between data generated by WGBS and methylation arrays. Swift achieved the highest proportion of CpG sites assayed and effective coverage at 26x (P < 0.001). TruSeq suffered from the highest proportion of PCR duplicates, while QIAseq failed to deliver across all quality metrics. There was little difference in performance between NovaSeq and HiSeq X, with the exception of higher read duplication rate on the NovaSeq (P < 0.05), likely attributable to the higher cluster densities on its flow cells. Systematic biases exist between WGBS and methylation arrays, with lower precision observed for WGBS across the range of depths investigated. To achieve a level of precision broadly comparable to the methylation array, a minimum coverage of 100x is recommended.

| S-EPMC6637168 | biostudies-literature

Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing [Beadchip]

Project description:We systematically evaluated alternate strategies for WGBS studies, taking into account opportunites around library preparation methods, sequencing platforms and analysis pipeline optimization. We also assessed the performance and precision of the WGBS method relative to the methylation arrays, in an effort to provide data-driven recommendations for future WGBS studies, in particularly with respect to minimum coverage.

2019-07-29 | GSE128733 | GEO

Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing [WGBS]

2019-07-29 | GSE128731 | GEO

Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing

Project description:Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing

| PRJNA528654 | ENA

Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing [WGBS]

Project description:Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing [WGBS]

| PRJNA528657 | ENA

Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing [Beadchip]

Project description:Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing [Beadchip]

| PRJNA528685 | ENA

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

Project description: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.

| S-EPMC4014543 | biostudies-literature

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing.

Project description:BACKGROUND:Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ?2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (? 2 nM) recommended by Illumina's protocols. RESULTS:Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with >?2 nM. CONCLUSIONS:A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.

| S-EPMC5935984 | biostudies-literature

Evaluation of a high-throughput, cost-effective Illumina library preparation kit.

Project description:Library preparation for high-throughput sequencing applications is a critical step in producing representative, unbiased sequencing data. The iGenomX Riptide High Throughput Rapid Library Prep Kit purports to provide high-quality sequencing data with lower costs compared to other Illumina library kits. To test these claims, we compared sequence data quality of Riptide libraries to libraries constructed with KAPA Hyper and NEBNext Ultra. Across several single-source genome samples, mapping performance and de novo assembly of Riptide libraries were similar to conventional libraries prepared with the same DNA. Poor performance of some libraries resulted in low sequencing depth. In particular, degraded DNA samples may be challenging to sequence with Riptide. There was little cross-well plate contamination with the overwhelming majority of reads belong to the proper source genomes. The sequencing of metagenome samples using different Riptide primer sets resulted in variable taxonomic assignment of reads. Increased adoption of the Riptide kit will decrease library preparation costs. However, this method might not be suitable for degraded DNA.

| S-EPMC8342411 | biostudies-literature

Systematic Evaluation of Medium-Throughput mRNA Abundance Platforms

Project description:Profiling of mRNA abundances with high-throughput platforms such as microarrays and RNA-Seq has become an important tool in both basic and biomedical research. However these platforms remain prone to systematic errors, and have challenges in clinical and industrial application. As a result it is standard practice to validate a subset of key results using alternate technologies. Similarly, clinical and industrial applications typically involve transitions from high-throughput discovery platform to medium-throughput validation ones. These medium-throughput validation platforms have high technical reproducibility and reduced sample input needs, and low sensitivity to sample-quality (e.g. for processing FFPE specimens). Unfortunately, while medium-throughput platforms have proliferated, there are no comprehensive comparisons of them. Here we present ABI's OpenArray and qPCR systems. TCDD-sensitive (Long-Evans) and TCDD-resistant (Han/Wistar) rats were used in both time-course and dose response studies to asses the transcriptomic response to TCDD-insult. The time-course experiment saw animals recieve a single dose of 100ug/kg TCDD or corn oil vehicle followed by euthanasia and tissue collection at various time-points. Animals in the dose-response experiment recieved a single dose of TCDD at varying concentrations or corn oil vehicle, followed by euthanasia and tissue collection 19 hours post-treatment. Each treatment group contained 4-5 biologicial replicates.

2012-10-18 | E-GEOD-41388 | biostudies-arrayexpress

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