HIGH THROUGHPUT GENOME-WIDE TRANSLOCATION SEQUENCING

• US 20140234847A1
• Filed: 07/06/2012
• Published: 08/21/2014
• Est. Priority Date: 07/07/2011
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation
• Original Assignee: Children's Medical Center Corporation
• Inventors: Alt, Frederick W., Zhang, Yu, Chiarle, Roberto, Gostissa, Monica
• Granted Patent: US 9,518,293 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/131 Inverse PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

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.

Claims

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;

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.

26. 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.

27. The method of claim 26, wherein the agent is a rare-cutting enzyme.

28. The method of claim 1, wherein the agent is a meganuclease, a TALEN or a zinc-finger nuclease.

29. The method of claim 1, wherein the cells are allowed to divide for 1-5 days.

30. The method of claim 28, wherein the cells are allowed to divide for 2-4 days.

31. The method of claim 1, wherein the sequencing is performed using a next generation sequencing method.

32. The method of claim 1, wherein the step of aligning is performed by a non-human machine.

33. The method of claim 32, wherein the non-human machine comprises a computer executable software.

34. The method of claim 33 further comprising a display module for displaying the results of the step of aligning.

35. The method of claim 1, wherein the cell is a mammalian cell.

36. The method of claim 1, wherein the cell is a plant cell.

2-25. -25. (canceled)

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

a. exposing a cell to an agent known or suspected to be capable of producing a 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 sample by ligating the fragmented DNA at a concentration favoring intra-molecular ligation;

f. digesting the ligated DNA sample with a blocking enzyme;

g. producing nested PCR products by performing a nested PCR with locus-specific primers;

h. sequencing the nested PCR products;

i. aligning the sequences against a reference sequence to identify chromosomal locations of the translocations and DSBs.

38. The method of claim 37, 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 37.

39. The method of claim 38, wherein the agent is a rare-cutting enzyme.

40. The method of claim 37, wherein the agent is a meganuclease, a TALEN or a zinc-finger nuclease.

41. The method of claim 37, wherein the cells are allowed to divide for 1-5 days.

42. The method of claim 41, wherein the cells are allowed to divide for 2-4 days.

43. The method of claim 37, wherein the sequencing is performed using a next generation sequencing method.

44. The method of claim 37, wherein the step of aligning is performed by a non-human machine.

45. The method of claim 44, wherein the non-human machine comprises a computer executable software.

46. The method of claim 45 further comprising a display module for displaying the results of the step of aligning.

47. The method of claim 37, wherein the cell is a mammalian cell.

48. The method of claims 37, wherein the cell is a plant cell.

49. The method of claim 37, wherein the cell division step (b) is omitted.