Measurement and comparison of immune diversity by high-throughput sequencing

Abstract

A precise measurement of the immunological receptor diversity present in a sample is obtained by sequence analysis. Samples of interest are generally complex, comprising more than 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or more different sequences for a receptor of interest. Immunological receptors of interest include immunoglobulins, T cell antigen receptors, and major histocompatibility receptors. The specific composition of immunological receptor sequence variations in the sample can be recorded and output. The composition is useful for predictive, diagnostic and therapeutic methods relating to the immune capabilities and history of an individual. Such predictions and diagnoses are used to guide clinical decisions.

1 .- 85 . (canceled) 
     
     
         86 . A method for characterizing a set of immunoglobulin receptors in a biological sample obtained from a subject exposed to an antigen, wherein the sample comprises a plurality of immunoglobulin heavy chain VDJ exon nucleic acid molecules, the method comprising,
 producing amplicons from the heavy chain VDJ exon sequences, wherein the amplicons are produced using a set of primers that amplify a plurality of immunoglobulin heavy chain VDJ exon sequences, wherein the amplicons comprise both the VD junction and the DJ junction;   sequencing the amplicons to obtain at least 10 4  sequence reads of immunoglobulin heavy chain sequences to obtain immunoglobulin heavy chain sequence information comprising sequences from a plurality of different genomic V segments, a plurality of different genomic D segments, and a plurality of different genomic J segments;   identifying heavy chain VDJ exon consensus sequences from the immunoglobulin heavy chain sequence information, wherein the heavy chain VDJ exon consensus sequences correspond to heavy chain VDJ exon sequences of the subject's immune repertoire,   forming single linkage clusters between the heavy chain VDJ exon consensus sequences, wherein a single linkage cluster comprises two heavy chain VDJ exon consensus sequences that vary by at most one nucleotide at a VDJ junction boundary, and have greater than or equal to 80% identity at the VDJ junction, wherein a VDJ junction is the exon region at the end of the V-encoded region to the beginning of the J-encoded region; and   chaining single clusters to form cluster lineages.   
     
     
         87 . The method of  claim 86 , wherein the heavy chain VDJ exon consensus sequences have reduced sequencing error compared to the immunoglobulin heavy chain sequence information. 
     
     
         88 . The method of  claim 86 , further comprising identifying the naïve heavy chain VDJ exon sequences of a cluster lineage by comparing the member sequences of the cluster lineage to reference genome, wherein the naïve heavy chain VDJ exon sequence is the heavy chain VDJ exon sequence that has the fewest differences relative to the reference genome. 
     
     
         89 . The method of  claim 88 , further comprising identifying mutations in the heavy chain VDJ exon consensus sequences by comparing the heavy chain VDJ exon consensus sequences to the naïve sequence. 
     
     
         90 . A method of comparing immunoglobulin repertoires of two or more subjects, comprising:
 characterizing a first subject's set of immunoglobulin receptors using the method of  claim 86 ; characterizing a second subject's set of immunoglobulin receptors using the method of claim  1 ; and comparing the characterization of the first subject's set of immunoglobulin receptors with the characterization of the second subject's set of immunoglobulin receptors.   
     
     
         91 . The method of  claim 86 , wherein the at least 10 4  sequence reads comprises at least 10 5 , 10 6 , 10 7 , 10 8 , 10 10 , 10 11  or 10 12  sequence reads. 
     
     
         92 . The method of  claim 86 , wherein the sample is blood, lymph, sputum or tissue. 
     
     
         93 . The method of  claim 86 , wherein the antigen is a cancer antigen, autoantigen, pathogenic antigen or a vaccine antigen. 
     
     
         94 . The method of  claim 86 , wherein the subject has or is suspected of having an autoimmune disorder, a symptom of allergy, asthma, an inflammatory disease, an infection, or cancer. 
     
     
         95 . The method of  claim 94 , wherein the cancer is prostrate, pancreas, colon, brain, lung, breast, bone, or skin cancer. 
     
     
         96 . The method of  claim 94 , wherein the inflammatory disease is irritable bowel syndrome, ulcerative colitis, appendicitis, tonsilitis, or dermatitis. 
     
     
         97 . The method of  claim 86 , wherein the antigen is a vaccine antigen or a pathogenic antigen. 
     
     
         98 . The method of  claim 86 , further comprising storing the immunoglobulin heavy chain sequence information on a computer-readable medium. 
     
     
         99 . The method of  claim 88 , wherein the reference genome comprises genomic V and J-segment sequences. 
     
     
         100 . The method of  claim 90 , wherein the comparing comprises obtaining a statistical metric of correlation between the two subjects' sets of immunoglobulin receptors.