# US 9,518,293 B2 · High throughput genome-wide translocation sequencing

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- Number: US 9,518,293 B2
- Document ID: US9518293B2
- Status: Granted
- Kind: B2
- Application number: 14/131,236
- Filed: 2012-07-06
- Published: 2014-08-21
- Granted: 2016-12-13
- Earliest claimed: 2011-07-07
- Assignees: Children's Medical Center Corporation
- Applicants: Frederick W. Alt; Zhang Yu; Roberto Chiarle; Monica Gostissa
- Inventors: Frederick W. Alt; Zhang Yu; Roberto Chiarle; Monica Gostissa
- Web page: https://patentgrep.com/patent/US9518293B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6855: Ligating adaptors
- C12Q 1/6869: Methods for sequencing
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## 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

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 2)

The method of claim 2, wherein the agent is a rare-cutting enzyme.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

The method of claim 1, wherein the cells are allowed to divide for 2-4 days.

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 7)

The method of claim 7, wherein the non-human machine comprises a computer executable software.

### Claim 9 (depends on 8)

The method of claim 8 further comprising a display module for displaying the results of the step of aligning.

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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