Multiplexed digital quantitation of rearranged lymphoid receptors in a complex mixture

A method for estimating an absolute abundance of individual T cell or B cell clones in a biological sample, comprising:

• A) distributing a biological sample from a human subject comprising T cells and/or B cells or DNA isolated from the T cells and/or B cells in the biological sample to a plurality of wells on a multi-well plate;
• B) amplifying rearranged T cell receptor (TCR) or immunoglobulin (IG) complementarity determining region 3 (CDR3) regions of the T cells and/or B cells in each well in a multiplex polymerase chain reaction (PCR) to obtain a first set of amplicons, wherein the multiplex PCR is performed with a plurality of V region specific primers and a plurality of J region specific primers such that the plurality of V region and J region specific primers amplify substantially all combinations of the V and J segments of the CDR3 regions of the T cells and/or B cells, and wherein the V region specific primers and/or the plurality of J region specific primers comprise a unique amplicon specific barcode;
• C) amplifying the first set of amplicons in a second PCR using tailing primers comprising a unique well-specific barcode for each well of the multi-well plate to obtain a second set of amplicons;
• D) sequencing by high-throughput sequencing the second set of amplicons to obtain a plurality of barcoded sequencing reads;
• E) identifying which well of the multi-well plate the barcoded reads are located in by identifying the unique well-specific barcodes on each of the sequencing reads;
• and
• F) estimating the absolute abundance of individual T cell or B cell clones having a particular TCR or IG-CDR3 region in the biological sample by application of likelihood (L) model equation (1) for probability P that w number of W total wells will contain a T cell or a B cell clone, wherein the w represents the number of wells containing a well-specific barcode, W represents the total number of wells in the multi-well plate, and n represents the total number of cells inputted into the wells:
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• wherein the absolute abundance of individual T cell or B cell clones in the sample reflects the human subject's immune status.