Measurement and comparison of immune diversity by high-throughput sequencing

Abstract

High-throughput long read sequencing is used to perform immunogenomic characterization of expressed antibody repertoires in the context of vaccination. Informatic analysis allows global characterizations of isotype distributions, determination of the lineage structure of the repertoire and measure age and antigen related mutational activity. Global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

1 .- 11 . (canceled) 
     
     
         12 . A method of identifying the presence of one or more antibody lineages in a sample, comprising,
 linking CDR3 nucleic acids present in the sample to establish a plurality of linked CDR3 nucleic acid sequences, wherein the CDR3 nucleic acid sequences are determined via high throughput sequencing of the nucleic acid in the sample, and wherein linked CDR3 nucleic acid sequences of the plurality comprise a first and second CR3 nucleic acid sequence having the same V and J assignment and no more than one amino acid difference in the CDR3 region; and   grouping CDR3 nucleic acid sequences into a plurality of lineages, wherein individual lineages of the plurality comprise (i) identical CDR3 nucleic acid sequences and (ii) the linked CDR3 sequences.   
     
     
         13 . The method of  claim 12 , wherein the sample is obtained from a subject exposed to a vaccine. 
     
     
         14 . The method of  claim 12 , further comprising quantitating the abundance of individual CDR3 amino acid sequences within individual lineages by counting the number of sequencing reads of the counterpart CDR3 nucleic acid sequences. 
     
     
         15 . The method of  claim 12 , further comprising,
 quantitating an average number of mutations within the individual lineages, wherein the average number of mutations is the average over a plurality of sequence reads of the CDR3 regions within the individual lineages.   
     
     
         16 . The method of  claim 12 , wherein the biological sample is selected from the group consisting of blood, lymph, sputum, and tissue. 
     
     
         17 . The method of  claim 16 , wherein the biological sample is blood. 
     
     
         18 . The method of  claim 12 , further comprising,
 (a) carrying out the method using a first biological sample obtained from the subject at a first time point;   (b) carrying out the method using a second biological sample obtained from the subject at a second time point; and   (c) comparing the lineage structure determined in (a) with the lineage structure determined in (b),   wherein after the time point and prior to the second time point the subject is exposed to an antigen.   
     
     
         19 . The method of  claim 12 , further comprising quantitating sizes of the individual lineages by determining the number of linked CDR3 sequences in the individual lineages. 
     
     
         20 . The method of  claim 12 , wherein the subject has an autoimmune disease. 
     
     
         21 . The method of  claim 20 , wherein the autoimmune disease is selected from the group consisting of insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), Crohn's disease, Graves' disease, celiac disease, and dermatitis herpetiformis.