Methods of nucleic acid sample preparation for immune repertoire sequencing

Abstract

Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids, e.g., nucleic acids encoding immune receptors and immunoglobulins. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein

1 - 68 . (canceled) 
     
     
         69 . A method of preparing nucleic acids for analysis, the method comprising:
 (a) contacting a nucleic acid molecule comprising a target nucleotide sequence with a first target-specific primer that specifically anneals to the target nucleotide sequence under hybridization conditions;   (b) conducting a first strand synthesis reaction that is primed by the hybridized first target-specific primer and that uses the nucleic acid molecule as a template, wherein a capture moiety is incorporated into a product of the first strand synthesis;   (c) conducting a second strand synthesis reaction that uses the product of the first strand synthesis reaction as a template to generate a double-stranded nucleic acid comprising the capture moiety;   (d) ligating an adapter nucleic acid to the double-stranded nucleic acid to produce a ligation product comprising the capture moiety;   (e) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety; and   (f) amplifying the captured ligation product by polymerase chain reaction using a second target-specific primer that comprises a 3′ portion that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a sequence of the adapter nucleic acid, wherein the second target-specific primer comprises a 5′ tail portion that does not specifically anneal to the target nucleotide sequence.   
     
     
         70 . The method of  claim 69 , wherein the first target-specific primer is a capture moiety modified primer comprising the capture moiety. 
     
     
         71 . The method of  claim 69 , wherein the conducting the first strand synthesis of (b) comprises incorporating one or more nucleotides into the product of the first strand synthesis, wherein the one or more nucleotides comprise the capture moiety. 
     
     
         72 . The method of  claim 69 , further comprising:
 (g) amplifying an amplification product of step (f) by polymerase chain reaction using a tail primer that comprises a 3′ portion that specifically anneals to a complementary sequence of the 5′ tail portion of the second target-specific primer and a second adapter primer that specifically anneals to a sequence of a portion of the adapter nucleic acid, wherein the tail primer comprises a 5′ portion that does not specifically anneal to a complementary sequence of the second target-specific primer.   
     
     
         73 . The method of  claim 69 , wherein:
 the adapter nucleic acid comprises a duplex portion; or   wherein the adapter nucleic acid is single-stranded.   
     
     
         74 . The method of  claim 69 , wherein:
 the adapter nucleic acid comprises a 5′ unpaired portion and a 3′ duplex portion; and   the first adapter primer specifically anneals to a complementary sequence of the 5′ unpaired portion.   
     
     
         75 . The method of  claim 69 , wherein the capture moiety comprises biotin, or a biotin moiety selected from biotin-triethylene glycol, bis-biotin, photocleavable biotin, desthiobiotin, desthiobiotin-triethylene glycol, and biotin azide. 
     
     
         76 . The method of  claim 69 , wherein the binding partner comprises streptavidin or streptavidin attached to a substrate. 
     
     
         77 . The method of  claim 69 , wherein the ligating of (d) is conducted in the presence of a crowding agent. 
     
     
         78 . The method of  claim 69 , wherein:
 the second strand synthesis reaction is primed by a fragment of the nucleic acid molecule hybridized to the product of the first strand synthesis reaction; or   the second strand synthesis is randomly primed using a plurality of random primers.   
     
     
         79 . The method of  claim 69 , wherein the nucleic acid molecule comprises mRNA. 
     
     
         80 . The method of  claim 69 , wherein the nucleic acid molecule is obtained from a sample comprising a T cell or a B cell. 
     
     
         81 . The method of  claim 80 , wherein:
 the sample is obtained from a subject having, or suspected of having, a T cell malignancy or a B cell malignancy;   the sample is obtained from a subject that has undergone or will undergo transplantation; or   the sample is obtained from a subject whose immune response to a treatment is being evaluated.   
     
     
         82 . The method of  claim 81 , wherein the subject is a human. 
     
     
         83 . The method of  claim 69 , wherein the target nucleotide sequence comprises a nucleotide sequence encoding a complementarity determining region of a T cell receptor (TCR), a B cell receptor (BCR) or an immunoglobulin. 
     
     
         84 . The method of  claim 83 , wherein:
 the first target-specific primer specifically anneals to a portion of the target nucleotide sequence corresponding to (i) a constant region, (i) a J-segment or (iii) an exon-exon junction formed between a constant region and a J-segment, of the T cell receptor gene, the B cell receptor gene or the immunoglobulin gene.   
     
     
         85 . The method of  claim 72 , wherein the first adapter primer and the second adapter primer are different. 
     
     
         86 . The method of  claim 72 , wherein the second adapter primer is nested relative to the first adapter primer and the second target-specific primer is nested relative to the first target-specific primer. 
     
     
         87 . The method of  claim 72 , wherein the 5′ portion of the tail primer comprises at least one of a sample index region, a molecular barcode region, and a sequencing primer site region. 
     
     
         88 . A method of preparing nucleic acids for analysis, the method comprising:
 (a) contacting a nucleic acid molecule comprising a target nucleotide sequence with a first target-specific primer that specifically anneals to the target nucleotide sequence under hybridization conditions;   (b) conducting a first strand synthesis reaction that is primed by the hybridized first target-specific primer and that uses the nucleic acid molecule as a template, and the conducting of the first strand synthesis comprises incorporating one or more nucleotides into a product of the first strand synthesis, wherein the one or more nucleotides comprise a capture moiety;   (c) conducting a second strand synthesis reaction that uses the product of the first strand synthesis reaction as a template to generate a double-stranded nucleic acid comprising the capture moiety;   (d) ligating an adapter nucleic acid to the double-stranded nucleic acid to produce a ligation product comprising the capture moiety;   (e) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety;   (f) amplifying the captured ligation product by a polymerase chain reaction using a second target-specific primer that comprises a 3′ portion that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a sequence of the adapter nucleic acid, wherein the second target-specific primer comprises a 5′ tail portion that does not specifically anneal to the target nucleotide sequence, thereby providing a first amplification product; and   (g) amplifying the first amplification product by a polymerase chain reaction using a tail primer that comprises a 3′ portion that specifically anneals to a complementary sequence of the 5′ tail portion of the second target-specific primer and a second adapter primer that specifically anneals to a portion of the adapter nucleic acid, wherein the tail primer comprises a 5′ portion that does not specifically anneal to a complementary sequence of the second target-specific primer.