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Status |
Public on Feb 21, 2012 |
Title |
Sequencing-based ssDNA detection |
Organism |
Mus musculus |
Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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Summary |
Meiotic DNA double stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. Although DSBs can be directly mapped using ChIP-Seq and antibody against ssDNA-associated proteins, 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 consists of 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. When applied to mapping meiotic DSBs, the use of SSDS reduces the non-specific dsDNA background more than ten-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.
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Overall design |
Development and validation of the method, SSDS, for the specific detection of ssDNA-derived and dsDNA-derived fragments in sequencing libraries and enrichment of ssDNA-derived fragments. SSDS was used to detect meiotic DSBs in 9R/13R mice.
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Contributor(s) |
Khil PP, Smagulova FO, Brick KM, Gregoretti IV, Camerini-Otero RD, Petukhova GV |
Citation(s) |
22367190 |
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Submission date |
Dec 20, 2011 |
Last update date |
May 15, 2019 |
Contact name |
Pavel P Khil |
E-mail(s) |
pk94j@nih.gov
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Phone |
301-496-4638
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Fax |
(301)-496-9878
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Organization name |
NIDDK
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Department |
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Lab |
Genetics and Biochemistry Branch
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Street address |
5 Memorial Dr.
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City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
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Platforms (2) |
GPL9250 |
Illumina Genome Analyzer II (Mus musculus) |
GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
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Samples (11)
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Relations |
SRA |
SRP009907 |
BioProject |
PRJNA150489 |