High throughput genome-wide translocation sequencing

A method for high throughput, genome-wide translocation sequencing (HTGTS) and detection of double-stranded DNA break (DSB) locations, the method comprising the steps of:

• a. exposing a cell to an agent known or suspected of being capable of producing at least one DSB;
• b. optionally allowing the cell to divide for at least 12 hours;
• c. extracting genomic DNA from the cells;
• d. producing a fragmented DNA sample by fragmenting the DNA of the cell with a frequently cutting restriction enzyme;
• e. producing a ligated DNA product by ligating an asymmetric adapter to the fragmented DNA sample, wherein the asymmetric adapter comprises a sequence that is designed to anneal to the DNA end generated by the frequently cutting restriction enzyme and contains a stretch of known DNA sequence that can be used to design a PCR primer for a nested PCR amplification;
• f. digesting the ligated DNA products with an enzyme to block amplification of germline or unrearranged targeted alleles;
• g. producing nested PCR products by performing nested-PCR with adapter- and locus-specific primers using the digested ligated DNA product thereby amplifying the nucleic acid sequences surrounding the junctions around the DSBs;
• h. producing sequenced nested PCR products by sequencing the nested PCR products;
• and
• i. aligning the sequenced nested PCR products against a reference sequence to identify chromosomal locations of the translocations and the chromosomal locations of the DSBs.