title: Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA.
abstract: 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.
authors.affiliation: Genetics and Biochemistry Branch, National Institute of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA.
authors.forename: Pavel P
authors.initials: PP
authors.lastname: Khil
authors.forename: Fatima
authors.initials: F
authors.lastname: Smagulova
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Brick
authors.forename: R Daniel
authors.initials: RD
authors.lastname: Camerini-Otero
authors.forename: Galina V
authors.initials: GV
authors.lastname: Petukhova
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D002874:Chromosome Mapping / Q000379:methods*; D053903:DNA Breaks, Double-Stranded; D004277:DNA, Single-Stranded / Q000235:genetics*; D015723:Gene Library; D055029:Inverted Repeat Sequences; D008297:Male; D008540:Meiosis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D004277:DNA, Single-Stranded