Methods and compositions relating to detection of recombination and rearrangement events

Abstract

Described herein are methods and assays for detection of recombination and/or rearrangement events in a cell. In some embodiments, the methods and/or assays relate to Linear Amplification Mediated (LAM)-PCR. In some embodiments, the recombination event is a V(D)J recombination event.

1 . A method for high throughput, genome-wide translocation sequencing (HTGTS)-based detection of recombination and/or rearrangement events in a cell, the method comprising the steps of:
 a. extracting genomic DNA and/or mRNA from a cell;   b. optionally, producing a fragmented DNA and/or mRNA sample;   c. producing:
 a single-stranded PCR product from genomic DNA by Linear Amplification Mediated (LAM)-PCR with at least one primary locus-specific primer; and/or 
 cDNA from mRNA by reverse-transcription with at least one primary locus-specific primer; 
   d. producing a ligated DNA and/or cDNA product by ligating the single-stranded PCR product or cDNA produced in step (c) to an adaptor, wherein the adaptor comprises:
 a distal portion of known DNA sequence that can be used to design PCR primers for a nested PCR amplification; 
 a proximal portion of random nucleotides; and 
 a 3′ overhang; 
   e. producing a nested PCR product by performing a nested-PCR with an adaptor-specific primer and at least one secondary locus-specific primer using the ligated product of step (d), thereby amplifying the nucleic acid sequence comprising the recombination and/or rearrangement event;   f. optionally, digesting the PCR product of step (e) with a restriction enzyme to blocks un-rearranged bait-containing fragments;   g. producing a sequenced nested PCR product by sequencing the nested PCR product; and   h. aligning the sequenced nested PCR product against a reference sequence or antigen receptor database.   
     
     
         2 . The method of  claim 1 , wherein the recombination event is a V(D)J recombination event. 
     
     
         3 . A method for high throughput, repertoire sequencing-based detection of Ig repertoire sequences in a cell, the method comprising the steps of:
 a. extracting genomic DNA and/or mRNA from a cell;   b. optionally, producing a fragmented DNA and/or mRNA sample;   c. producing:
 a single-stranded PCR product from genomic DNA by Linear Amplification Mediated (LAM)-PCR with at least one primary locus-specific primer; and/or 
 cDNA from mRNA by reverse-transcription with at least one primary locus-specific primer; 
   d. producing a ligated DNA and/or cDNA product by ligating the single-stranded PCR product or cDNA produced in step (c) to an adaptor, wherein the adaptor comprises:
 a distal portion of known DNA sequence that can be used to design PCR primers for a nested PCR amplification; 
 a proximal portion of random nucleotides; and 
 a 3′ overhang; 
   e. producing a nested PCR product by performing a nested-PCR with an adaptor-specific primer and at least one secondary locus-specific primer using the ligated product of step (d), thereby amplifying the nucleic acid sequence comprising the Ig repertoire sequence;   f. optionally, digesting the PCR product of step (e) with a restriction enzyme to block un-rearranged bait-containing fragments;   g. producing a sequenced nested PCR product by sequencing the nested PCR product; and   h. aligning the sequenced nested PCR product against a reference sequence or antigen receptor database.   
     
     
         4 . The method of  claim 3 , wherein the repertoire detected comprises V(D)J recombination events and/or somatic hypermutations (SMH). 
     
     
         5 . The method of  claim 3 , wherein the repertoire detected comprises Ig heavy chains, Ig light chains, V usage, and CDR3 repetoires. 
     
     
         6 . The method of  claim 1 , wherein the cell is selected from a group consisting of:
 a mature B lymphocyte, a developing B lymphocyte, a mature T lymphocyte, a developing T lymphocyte, a cell obtained from a germinal center, and a cell obtained from a Peyer's Patch.   
     
     
         7 . The method of  claim 1 , wherein the method further comprises providing the cell, wherein the cell was obtained from an animal immunized with an antigen. 
     
     
         8 . The method of  claim 1 , wherein the method further comprises providing the cell, wherein the cell comprises a V(D)J exon which has undergone somatic hypermutation. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the at least one primary locus-specific primer specifically anneals to J gene segments. 
     
     
         12 . The method of  claim 1 , wherein the method further comprises the use of multiple primary locus-specific primers and/or secondary locus-specific primers. 
     
     
         13 . The method of  claim 12 , wherein each of the multiple primers specifically anneal to different V, D, and/or J gene segments. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 13 , wherein, collectively, the multiple primers specifically anneal i) to a sequence in each of J H 1, J H 2, J H 3, or J H 4 or ii) to at least one sequence in each of the J H , J K , and J L  gene segments present in the genome of the cell or organism prior to V(D)J recombination. 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 1 , wherein the at least one primary locus-specific primer specifically anneals to a degenerate region(s) of the targeted gene segment(s). 
     
     
         18 . The method of  claim 1 , further comprising a step of differentiating a source cell or tissue to initiate V(D)J recombination prior to performing step (a). 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , further comprising a step of contacting the cell with one or more reagents that initiate V(D)J recombination or SHM. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein the rearrangement event involves an oncogene and/or a RAG off-target cutting site. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the primary locus-specific primer comprises an affinity tag. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 1 , wherein the primers used for the nested PCR step comprise barcode sequences. 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 1 , wherein ligating the product of step (c) to an adaptor comprises contacting the product with a population of adaptors having the same distal portion and random proximal portion sequences. 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 1 , wherein the adaptor comprises barcode sequences between distal and proximal portions. 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . The method of  claim 1 , wherein the blocking digestion step (f) is omitted. 
     
     
         49 . The method of  claim 1 , wherein end repair is not performed prior to step (c). 
     
     
         50 . The method of  claim 1 , wherein one or more of the primers comprises a sequence selected from SEQ ID Nos: 1-32 or 43-65. 
     
     
         51 . (canceled)