METHODS OF SEQUENCING THE IMMUNE REPERTOIRE
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 . 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,
b. producing immunoglobulin chain or TCR chain cDNAs from the RNA;
c. adding a homopolymeric tail, a random molecular tag and a universal sequence to the 3′ end of the cDNAs;
d. amplifying the cDNAs by using a one or more immunoglobulin chain or TCR chain specific primers and a universal sequence specific primer comprising an flanking sequence specific to the sequencing platform to produce a plurality of molecular tagged immunoglobulin chain or TCR chain nucleic acids;
e. sequencing the amplified immunoglobuling chain or TCR chain nucleic acids to produce a plurality of sequencing reads thereby determining the immune repertoire of the subject.
2 . The method of claim 1 , further comprising a data analysis step after step (e).
3 . The method of claim 2 , wherein the data analysis includes grouping sequences reads with the same molecular tag and clustering sequences within the same group.
4 . The method of claim 3 , further comprising building a consensus sequence for each cluster to produce a collection of consensus sequences.
5 . The method of claim 4 , wherein the collection of consensus sequences is used to determine the diversity of the immune repertoire.
6 . The method of claim 1 , wherein the molecular tag is an oligomer.
5 . The method of claim 6 , wherein the oligomer is at least a 9mer.
7 . The method of claim 1 , wherein the biological sample is blood or a fraction thereof.
8 . The method of claim 7 , wherein the blood is peripheral whole blood.
9 . The method of claim 7 , wherein the blood fraction comprises peripheral blood mononuclear cells.
10 . The method of claim 7 , wherein the blood sample is sorted based upon extracellular or intracellular markers.
11 . The method of claim 1 , wherein the immunoglobulin chain is the heavy chain or the light chain.
12 . The method of claim 11 , wherein immunoglobulin heavy chain comprise the immunoglobulin VDJ and constant regions.
13 . The method of claim 1 , wherein the TCR chain is the alpha chain, the beta chain, the gamma chain or the delta chain.
14 . The method of claim 13 , wherein the TCR chain comprises the VJ and constant regions.