Methods of sequencing the immune repertoire

Abstract

The invention provides a non-invasive technique for the detection and quantification of the immune repertoire, in a biological sample containing a plurality of distinct cell populations. Methods are conducted using sequencing technology to detect and enumerate immune repertoire within a heterogeneous biological sample.

1 .- 15 . (canceled) 
     
     
         16 . A method of determining the immune repertoire in a subject, comprising:
 (a) isolating a plurality of RNA from a biological sample comprising a plurality of cell types obtained from a subject, wherein the RNA comprises immunoglobulin chain mRNAs;   (b) reverse transcribing the mRNAs to produce immunoglobulin chain cDNAs comprising a 3′ homopolymer tail by using immunoglobulin chain specific primers and a reverse transcriptase;   (c) adding a random molecular tag and a universal sequence to the 3′ end of the immunoglobulin chain cDNAs to form extended cDNAs, by using the reverse transcriptase and oligonucleotides comprising a 3′ sequence complementary to the homopolymer tail and a 5′ flanking sequence comprising the universal sequence and the random molecular tag, wherein each extended cDNA is associated with a unique molecular tag;   (d) amplifying the extended cDNAs by using one or more immunoglobulin chain specific primers and a primer specific to the universal sequence and further comprising a sequencing platform specific flanking sequence to produce a plurality of molecular tagged immunoglobulin chain nucleic acids; and   (e) sequencing the molecular tagged immunoglobulin chain nucleic acids to produce a plurality of sequencing reads thereby determining the immune repertoire of the subject.   
     
     
         17 . The method of  claim 16 , further comprising a data analysis step after step (e). 
     
     
         18 . The method of  claim 17 , wherein the data analysis comprises grouping sequencing reads with the same molecular tag and clustering sequences within the same group to form clusters. 
     
     
         19 . The method of  claim 18 , further comprising building a consensus sequence for each cluster to produce a collection of consensus sequences. 
     
     
         20 . The method of  claim 19 , wherein the collection of consensus sequences is used to determine the diversity of the immune repertoire. 
     
     
         21 . The method of  claim 16 , wherein the molecular tag is an oligomer. 
     
     
         22 . The method of  claim 16 , wherein the biological sample is blood or a fraction thereof. 
     
     
         23 . The method of  claim 22 , wherein the blood is peripheral whole blood. 
     
     
         24 . The method of  claim 22 , wherein the blood fraction comprises peripheral blood mononuclear cells. 
     
     
         25 . The method of  claim 22 , wherein the blood sample is sorted based upon extracellular or intracellular markers. 
     
     
         26 . The method of  claim 16 , wherein the immunoglobulin chain is the heavy chain or the light chain. 
     
     
         27 . The method of  claim 26 , wherein the immunoglobulin heavy chain comprises the immunoglobulin VDJ and constant regions. 
     
     
         28 . The method of  claim 21 , wherein the oligomer is at least a 9 mer. 
     
     
         29 . A method of determining the immune repertoire in a subject, comprising:
 reverse transcribing mRNAs from a biological sample, wherein the mRNAs comprise immunoglobulin chain mRNAs, to produce immunoglobulin chain cDNAs comprising a 3′ homopolymer tail by using immunoglobulin chain specific primers and a reverse transcriptase;   adding a random molecular tag and a universal sequence to the 3′ end of the immunoglobulin chain cDNAs to form extended cDNAs, by using the reverse transcriptase and oligonucleotides comprising a 3′ sequence complementary to the homopolymer tail and a 5′ flanking sequence comprising the universal sequence and the random molecular tag, wherein each extended cDNA is associated with a unique molecular tag;   amplifying the extended cDNAs by using one or more immunoglobulin chain specific primers and a primer specific to the universal sequence and further comprising a sequencing platform specific flanking sequence to produce a plurality of molecular tagged immunoglobulin chain nucleic acids;   sequencing the molecular tagged immunoglobulin chain nucleic acids to produce a plurality of sequencing reads, thereby determining the immune repertoire of the subject.   
     
     
         30 . The method of  claim 29 , wherein the immunoglobulin chain is the immunoglobulin heavy chain or the light chain. 
     
     
         31 . The method of  claim 30 , wherein the immunoglobulin heavy chain comprises the immunoglobulin VDJ and constant regions. 
     
     
         32 . The method of  claim 29 , wherein the molecular tag is an oligomer and is at least a 9 mer.