MULTIPLEXED DIGITAL QUANTITATION OF REARRANGED LYMPHOID RECEPTORS IN A COMPLEX MIXTURE

摘要

The invention relates to methods and compositions for estimating the absolute abundance individually for each unique rearranged lymphocyte receptor in a mixed sample.

主权项

1. A method for estimating the absolute abundance of individual T cell or B cell clones in a complex mixture of cells comprising:
A) distributing a sample comprising T cells and/or B cells or DNA isolated from a sample of T cells and/or B cells among 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 plurality of amplicons; 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 plurality of barcoded amplicons; D) sequencing said barcoded amplicons to obtain a plurality of barcoded sequencing reads; E) identifying which well of the multi-well plate the barcoded sequence reads are located in by identifying the well-specific barcodes on each of the sequencing reads; and F) using a likelihood model to infer the most likely number of cells having a particular TCR or IG sequence in the starting sample based on the probability that any particular number of input cells would be present in the same number of wells as the observed TCR and IG sequence.