Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA

• Published in: Genome research Vol. 22; no. 5; pp. 957 - 965
• Main Authors: Khil, Pavel P, Smagulova, Fatima, Brick, Kevin M, Camerini-Otero, R Daniel, Petukhova, Galina V
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01-05-2012
• Subjects: Animals; Biochemistry, Molecular Biology; Chromatin; Chromatin Immunoprecipitation; Chromosome Mapping; Chromosome Mapping - methods; Computer applications; DNA Breaks, Double-Stranded; DNA damage; DNA sequencing; DNA, Single-Stranded; DNA, Single-Stranded - genetics; Gene Library; Gene mapping; Genomes; Germ cells; Hot spots; Immunoprecipitation; Inverted Repeat Sequences; Life Sciences; Male; Meiosis; Meiosis - genetics; Method; Mice; Mice, Inbred C57BL; Recombination; Recombination, Genetic; Sequence Analysis, DNA
• ISSN: 1088-9051; 1549-5469
• DOI: 10.1101/gr.130583.111

Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method--single-stranded DNA (ssDNA) sequencing (SSDS)--that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.