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Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm.

ABSTRACT: Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity. These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers. We show that chromatin environment on the template homolog affects positioning of crossover breakpoints. Our results also offer insights into recombination in the pseudoautosomal region.

SUBMITTER: Hinch AG

PROVIDER: S-EPMC6445350 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm.

Hinch Anjali Gupta AG Zhang Gang G Becker Philipp W PW Moralli Daniela D Hinch Robert R Davies Benjamin B Bowden Rory R Donnelly Peter P

Science (New York, N.Y.) 20190301 6433

Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-init ...[more]

PMID: 30898902

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Project description:Next-generation sequencing (NGS) technology has paved the way for rapid and cost-efficient de novo sequencing of bacterial genomes. In particular, the introduction of PCR-free library preparation procedures (LPPs) lead to major improvements as PCR bias is largely reduced. However, in order to facilitate the assembly of Illumina paired-end sequence data and to enhance assembly performance, an increase of insert sizes to facilitate the repeat bridging and resolution capabilities of current state of the art assembly tools is needed. In addition, information concerning the relationships between genomic GC content, library insert size and sequencing quality as well as the influence of library insert size, read length and sequencing depth on assembly performance would be helpful to specifically target sequencing projects.Optimized DNA fragmentation settings and fine-tuned resuspension buffer to bead buffer ratios during fragment size selection were integrated in the Illumina TruSeq(®) DNA PCR-free LPP in order to produce sequencing libraries varying in average insert size for bacterial genomes within a range of 35.4-73.0 % GC content. The modified protocol consumes only half of the reagents per sample, thus doubling the number of preparations possible with a kit. Examination of different libraries revealed that sequencing quality decreases with increased genomic GC content and with larger insert sizes. The estimation of assembly performance using assembly metrics like corrected NG50 and NGA50 showed that libraries with larger insert sizes can result in substantial assembly improvements as long as appropriate assembly tools are chosen. However, such improvements seem to be limited to genomes with a low to medium GC content. A positive trend between read length and assembly performance was observed while sequencing depth is less important, provided a minimum coverage is reached.Based on the optimized protocol developed, sequencing libraries with flexible insert sizes and lower reagent costs can be generated. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes.

Dataset Information

Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm.

Publications

Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm.

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