High throughput genome-wide translocation sequencing
Provided are methods for high-throughput screening to determine locations of double-stranded DNA breaks (DSBs) and translocations in genomes caused by different agents, such as enzymes.
1. 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.
2. The method of claim 1 , further comprising a step of inserting into a cell to be analyzed at least one target sequence for the agent that is known to be absent in the genome of the cell to be analyzed prior to step (a) of claim 1 .
3. The method of claim 2 , wherein the agent is a rare-cutting enzyme.
4. The method of claim 1 , wherein the agent is a meganuclease, a transcription activator-like effector nuclease which are sequence specific nucleases made by fusing a transcription activator like effector DNA-binding domain to a catalytic domain of an endonuclease or a zinc-finger nuclease.
5. The method of claim 1 , wherein the cells are allowed to divide for 1-5 days.
6. The method of claim 1 , wherein the cells are allowed to divide for 2-4 days.
7. The method of claim 1 , wherein the step of aligining is performed by a non-human machine.
8. The method of claim 7 , wherein the non-human machine comprises a computer executable software.
9. The method of claim 8 further comprising a display module for displaying the results of the step of aligning.
10. The method of claim 1 , wherein the cell is a mammalian cell.
11. The method of claim 1 , wherein the cell is a plant cell.