Dataset Information

Genotyping 1000 yeast strains by next-generation sequencing.

ABSTRACT:

Background

The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups.

Results

Up to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes.

Conclusions

Here we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.

SUBMITTER: Wilkening S

PROVIDER: S-EPMC3575377 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Publications

Genotyping 1000 yeast strains by next-generation sequencing.

Wilkening Stefan S Tekkedil Manu M MM Lin Gen G Fritsch Emilie S ES Wei Wu W Gagneur Julien J Lazinski David W DW Camilli Andrew A Steinmetz Lars M LM

BMC genomics 20130209

<h4>Background</h4>The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapte ...[more]

PMID: 23394869

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Project description:BackgroundHIV genotyping has had a significant impact on the care and treatment of HIV/AIDS. At a clinical level, the test guides physicians on the choice of treatment regimens. At the surveillance level, it informs policy on consolidated treatment guidelines and microbial resistance control strategies. Until recently, the conventional test has utilized the Sanger sequencing (SS) method. Unlike Next Generation Sequencing (NGS), SS is limited by low data throughput and the inability of detecting low abundant drug-resistant variants. NGS can improve sensitivity and quantitatively identify low-abundance variants; in addition, it has the potential to improve efficiency as well as lowering costs when samples are batched. Despite the NGS benefits, its utilization in clinical drug resistance profiling is faced with mixed reactions. These are largely based on a lack of a consensus regarding the quality control strategy. Nonetheless, transitional views suggest validating the method against the gold-standard SS. Therefore, we present a validation report of an NGS-based in-house HIV genotyping method against the SS method in Uganda.ResultsSince there were no established proficiency test panels for NGS-based HIV genotyping, 15 clinical plasma samples for routine care were utilized. The use of clinical samples allowed for accuracy and precision studies. The workflow involved four main steps; viral RNA extraction, targeted amplicon generation, amplicon sequencing and data analysis. Accuracy of 98% with an average percentage error of 3% was reported for the NGS based assay against the SS platform demonstrating similar performance. The coefficient of variation (CV) findings for both the inter-run and inter-personnel precision showed no variability (CV ≤ 0%) at the relative abundance of ≥ 20%. For both inter-run and inter-personnel, a variation that affected the precision was observed at 1% frequency. Overall, for all the frequencies, CV registered a small range of (0-2%).ConclusionThe NGS-based in-house HIV genotyping method fulfilled the minimum requirements that support its utilization for drug resistance profiling in a clinical setting of a low-income country. For more inclusive quality control studies, well-characterized wet panels need to be established.

Dataset Information

Genotyping 1000 yeast strains by next-generation sequencing.

Background

Results

Conclusions

Publications

Genotyping 1000 yeast strains by next-generation sequencing.

Similar Datasets

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