# US 10,246,701 B2 · Multiplexed digital quantitation of rearranged lymphoid receptors in a complex mixture

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,246,701 B2
- Document ID: US10246701B2
- Status: Granted
- Kind: B2
- Application number: 14/941,295
- Filed: 2015-11-13
- Published: 2016-05-19
- Granted: 2019-04-02
- Earliest claimed: 2014-11-14
- Assignees: Adaptive Biotechnologies Corporation
- Applicants: Adaptive Biotechnologies Corporation
- Inventors: William S Dewitt; Ryan O Emerson; Harlan S Robins; Anna M Sherwood
- Web page: https://patentgrep.com/patent/US10246701B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6827: for detection of mutation or polymorphism
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

The method of claim 1, wherein the DNA isolated from the sample of T cells and/or B cells is genomic DNA.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 5)

The method of claim 5, wherein the tailing primers further comprise a sequencing adaptor oligonucleotide sequence.

### Claim 7 (depends on 1)

The method of claim 1, wherein the unique amplicon specific barcode is used to determine carry over contamination between the wells.

### Claim 8 (depends on 7)

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.

### Claim 9 (depends on 1)

The method of claim 1, wherein the multi-well plate is a 96 well plate.