Enhanced immune cell receptor sequencing methods

Abstract

Disclosed are methods for sequencing immune cell receptor repertoires from immune cell populations, the methods comprising isolating RNA from immune cells, generating cDNA from the RNA, ligating adapter sequences to the cDNA, and sequencing the cDNA. Also provided are kits containing primer mixtures for the sequencing of immune cell receptor repertoires.

1 .- 30 . (canceled) 
     
     
         31 . A method for sequencing immune cell receptor genes, comprising providing RNA from immune cells;
 (a) (1) transcribing the RNA into complementary RNA (cRNA), using one or more primers wherein the cRNA is produced by transcription;
 (2) reverse transcribing the cRNA into single strand DNA (ssDNA), using one or more primers that comprise a 5′ adapter sequence, wherein each 5′ end of the ssDNA produced by reverse transcription contains the 5′ adapter sequence; 
   or   (b) (1) transcribing the RNA into complementary RNA (cRNA), using one or more primers that comprise a 3′ adapter sequence, wherein each 3′ end of the cRNA produced by transcription contains the 3′ adapter sequence;
 (2) reverse transcribing the cRNA into single strand DNA (ssDNA), using one or more primers that comprise a 5′ adapter sequence, wherein each 5′ end of the ssDNA produced by reverse transcription contains the 5′ adapter sequence and wherein each 3′ end of the ssDNA produced by reverse transcription contains the 3′ adapter sequence; 
   amplifying the ssDNA to produce a first amplification product using a first primer pair comprising a first primer that hybridizes to the 5′ adapter sequence and a second primer that hybridizes to a constant region of immune cell receptor gene or to the 3′ adapter sequence;   amplifying the first amplification product to produce a second amplification product using a second primer pair, in which
 i. a first primer of the second primer pair binds to the adapter sequence at the 5′ end of the first amplification product, 
 ii. the second primer of the second primer pair binds to the constant region of immune cell receptor gene in the first amplification product, and 
 iii. the first and second primers comprise adapter sequences for sequencing; and sequencing the second amplification product. 
   
     
     
         32 . The method according to  claim 31 , wherein the reverse transcription step results in PCR products ranging from 150-600 bp. 
     
     
         33 . The method according to  claim 31 , wherein the immune cell receptor genes are T-cell receptor (TCR) genes or B-cell receptor (BCR) genes. 
     
     
         34 . The method according to  claim 31 , wherein the one or more primers used for reverse transcription hybridize to TCR α chain V segments, optionally wherein the one or more primers used for reverse transcription comprise one or more of SEQ ID NOs: 1-50; or
 wherein the one or more primers used for reverse transcription hybridize to TCR β chain V segments, optionally wherein the one or more primers used for reverse transcription comprise one or more of SEQ ID NOs: 51-100; or 
 wherein the one or more primers used for reverse transcription hybridize to TCR γ chain V segments; or 
 wherein the one or more primers used for reverse transcription hybridize to TCR β chain V segments; or 
 wherein the one or more primers used for reverse transcription hybridize to BCR heavy chain V segments; or 
 wherein the one or more primers used for reverse transcription hybridize to BCR light chain V segments. 
 
     
     
         35 . The method according to  claim 31 , wherein the one or more primers used for reverse transcription contain a nucleotide barcode sequence; optionally wherein the nucleotide barcode comprises 6 to 20 nucleotides. 
     
     
         36 . The method according to  claim 35 , wherein the nucleotide barcode consists of 9 nucleotides; optionally wherein the nucleotide barcode consists of the sequence NNNNTNNNN, NNNNANNNN or HHHHHNNNN. 
     
     
         37 . The method according to  claim 31 , wherein the 5′ adapter sequence of the one or more primers used for the reverse transcription comprises a T7 adapter. 
     
     
         38 . The method according to  claim 31 , wherein the immune cells are T-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a TCR gene. 
     
     
         39 . The method according to  claim 31 , wherein the immune cells are B-cells and wherein the second primer of the first pair of primers hybridizes to the constant region of a BCR gene. 
     
     
         40 . The method according to  claim 31 , wherein the sequencing is next generation sequencing. 
     
     
         41 . The method according to  claim 31 , wherein the RNA from the immune cells is obtained by mixing immune cells with carrier cells before RNA extraction. 
     
     
         42 . The method according to  claim 31 , wherein the immune cells are tumor-infiltrating lymphocytes. 
     
     
         43 . The method according to  claim 31 , wherein the immune cells are CD4 or CD8 positive T-cells. 
     
     
         44 . The method according to  claim 31 , wherein the immune cells are purified from peripheral blood mononuclear cells (PBMC) before RNA extraction. 
     
     
         45 . The method according to  claim 31 , wherein the immune cells are part of a mixture of peripheral blood mononuclear cells (PBMC). 
     
     
         46 . The method according to  claim 31 , wherein the immune cells are derived from a mammal; optionally wherein the mammal is a human or a mouse. 
     
     
         47 . A kit for sequencing of T-cell receptors (TCRs), comprising:
 (a) at least one primer which comprises a TCR α chain V segment and a barcode sequence; or   (b) at least one primer which comprises a TCR β chain V segment and a barcode sequence.   
     
     
         48 . The kit according to  claim 47 ,
 (a) wherein the at least one primer which comprises a TCR α chain V segment and a barcode sequence comprises any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310; or   (b) wherein the at least one primer which comprises a TCR β chain V segment and a barcode sequence comprises any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.   
     
     
         49 . The kit according to  claim 48 , wherein the kit comprises at least one primer which comprises a sequence of any one of SEQ ID NOs: 1-50 or SEQ ID NOs: 261-310. 
     
     
         50 . The kit according to  claim 48 , wherein the kit comprises at least one primer which comprises a sequence of any one of SEQ ID NOs: 51-100 or SEQ ID NOs: 311-360.