Multiplexed digital quantitation of rearranged lymphoid receptors in a complex mixture
The invention relates to methods and compositions for estimating the absolute abundance individually for each unique rearranged lymphocyte receptor in a mixed sample.
1. 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:
ℒ
=
P
(
N
wells
(
obs
)
=
w
|
N
cells
=
n
,
N
wells
(
tot
)
=
W
)
=
W
!
{
n
w
}
(
W
-
w
)
!
W
n
,
(
Equation
1
)
wherein the absolute abundance of individual T cell or B cell clones in the sample reflects the human subject's immune status.
2. The method of claim 1 , wherein the biological sample comprising the T cells and/or B cells is a blood sample or a lymph sample.
3. The method of claim 1 , wherein the DNA isolated from the sample of T cells and/or B cells is genomic DNA.
4. The method of claim 1 , wherein the plurality of V region specific primers and plurality of J region specific primers further comprise a universal adaptor sequence.
5. The method of claim 4 , wherein the tailing primers comprise a sequence complementary to the universal adaptors added to the first set of amplicons in step B.
6. The method of claim 5 , wherein the tailing primers further comprise a sequencing adaptor oligonucleotide sequence.
7. The method of claim 1 , wherein the unique amplicon specific barcode is used to determine carry over contamination between the wells.
8. The method of claim 7 , wherein the carry over contamination is detected by the presence of the same sequence read with the same unique amplicon specific barcode in multiple wells.
9. The method of claim 1 , wherein the multi-well plate is a 96 well plate.