Quantification of adaptive immune cell genomes in a complex mixture of cells

Patent metadata

Field	Value
Publication number	US-10150996-B2
Application number	US-201515304631-A
Country	US
Kind code	B2
Filing date	Mar 5, 2015
Priority date	Oct 19, 2012
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

A relative representation of adaptive immune cells in a biological sample is quantified using multiplex PCR and sequencing of adaptive immune cells, control genes, and synthetic template molecules. Disclosed herein are methods for quantifying a number of adaptive immune cells in a biological sample, and methods for quantifying a relative representation of adaptive immune cells in a biological sample that comprises a mixture of cells comprising adaptive immune cells and cells that are not adaptive immune cells. Methods are provided for amplifying by multiplex PCR and sequencing a first set of synthetic templates each comprising one TCR or IgV segment and one TCR of Ig J or C segment and a unique bar code.

First claim

The invention claimed is:

1. A method for determining a ratio of T cells or B cells in a sample relative to the total number of input genomes contained in said sample comprising:
A) amplifying by multiplex PCR and sequencing:
i) rearranged CDR3 oligonucleotide sequences from T cell receptor (TCR) loci from T cells or Immunoglobulin (Ig) loci from B cells in said sample to obtain a total number of output biological sequences, each oligonucleotide sequence comprising a V segment and a J segment;
ii) a first set of synthetic templates representing substantially all possible V segment and J segment combinations and each comprising one TCR or Ig V segment and one TCR or Ig J or C segment and a unique barcode which identifies said synthetic template as synthetic, and wherein each synthetic template comprises a unique combination of a V segment and J or C segment;

B) determining an amplification factor for each synthetic template comprising a unique combination of a V segment and a J or C segment, wherein said amplification factor is represented by a total number of first synthetic templates observed from step A(ii) divided by a total input number of unique first synthetic templates input in step A(ii);
C) determining the total number of T cells or B cells in the sample by dividing the total number of output biological sequences observed in step A(i) by the amplification factor from step B;
D) amplifying by multiplex PCR and sequencing:
i) one or more genomic control regions from DNA obtained from said sample to obtain a total number of output biological sequences for each genomic control region; and
ii) a second set of synthetic templates comprising the sequence of one or more of said genomic control sequences, a unique barcode and a stretch of random nucleic acids, wherein each synthetic template is represented only once;
E) determining an amplification factor for each of said genomic control region by dividing the total number of second synthetic templates amplified and sequenced in step D(ii) by the total input number of unique second synthetic templates amplified and sequenced in step D(ii);
F) determining the total number of input genomes by dividing the total number of output biological sequences for each genomic control region from step D(i) by the corresponding amplification factor for that genomic control region from Step E; and
G) determining the ratio of T cells or B cells contained in the sample relative to the number of total genomes in the sample by dividing the total number of T cells or B cells obtained in step C by the total number of input genomes obtained in step F.

2. The method of claim 1 , wherein the first set of synthetic templates comprises the sequence of formula I: 5′-U1-B1-V-B2-J-B3-U2-3′, wherein
A) V is an oligonucleotide sequence comprising at least 20 and not more than 1000 contiguous nucleotides of a TCR or Ig variable (V) region encoding gene sequence, or the complement thereof and each template in set first set of synthetic templates having a unique V-region oligonucleotide sequence;
B) J is an oligonucleotide sequence comprising at least 15 and not more than 600 contiguous nucleotides of a TCR or Ig joining (J) region encoding gene sequence, or the complement thereof and each template in said first set of synthetic templates comprising a unique J-region oligonucleotide sequence;
C) U1 comprises an oligonucleotide sequence that is selected from (i) a first universal adaptor oligonucleotide sequence; and (ii) a first sequencing platform oligonucleotide sequence that is linked to and positioned 5′ to a first universal adaptor oligonucleotide sequence;
D) U2 comprises an oligonucleotide sequence that is selected from (i) a second universal adaptor oligonucleotide sequence; and (ii) a second sequencing platform oligonucleotide sequence that is linked to and positioned 5′ to a second universal adaptor oligonucleotide sequence; and
E) B1, B2 and B3 each independently comprise either nothing or an oligonucleotide barcode sequence of 3-25 nucleic acids that uniquely identifies, as a pair combination (i) said unique V-region oligonucleotide sequence; and said unique J-region oligonucleotide, wherein at least one of B1, B2 and B3 is present in each synthetic template contained in said first set of oligonucleotides.

3. The method of claim 2 , wherein each of the synthetic templates contained in said first set of synthetic templates further comprises a stretch of unique random nucleotides.

4. The method of claim 3 , wherein the random stretch of nucleotides comprise from 4 to 50 nucleotides, or wherein the random stretch of nucleotides comprises 8 nucleotides.

5. The method of claim 1 , wherein the total number of synthetic templates in said first set of synthetic templates subject to amplification in step A(ii) is determined using a limiting dilution of said synthetic templates each comprising a unique TCR or Ig V and J or C region such that the number of observed unique synthetic templates allows inference of the total number of input synthetic template molecules.

6. The method of claim 3 , wherein the total number of synthetic templates in said first set of synthetic templates subject to amplification in step A(ii) is determined by counting the number of unique synthetic templates based on the unique random nucleotides contained in each synthetic template.

7. The method of claim 1 , comprising amplifying by multiplex PCR and sequencing two or more, or three or more, or four or more, or five or more genomic control regions in step D(i).

8. The method of claim 1 , comprising amplifying by multiplex PCR and sequencing five genomic control regions in step D(i), and wherein each of the five genomic control regions has a predictable copy number.

9. The method of claim 1 , wherein the one or more genomic control regions are selected from the group consisting of ACTB, B2M, C1orf34, CHMP2A, GPI, GUSB, HMBS, HPRT1, PSMB4, RPL13A, RPLP0, SDHA, SNRPD3, UBC, VCP, VSP29, PPIA, PSMB2, RAB7A, REEP5, and EMC7, or wherein the one or more genomic control regions are PSMB2, RAB7A, PPIA, REEP5, and EMC7.

10. The method of claim 8 , wherein amplification factors are determined for each of said five genomic control regions in step E.

11. The method of claim 10 , wherein the total number of input genomes is calculated in step F by taking an average using each of the five amplification factors determined for each of said five genomic control regions.

12. The method of claim 1 , wherein the amplification by multiplex PCR in steps A and D are done in a single multiplex reaction.

13. The method of claim 1 , wherein the amplification by multiplex PCR in step A or step D is performed using a plurality of oligonucleotide primer sets comprising:
A) a plurality of V segment oligonucleotide primers that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a TCR of Ig V region polypeptide or to the complement thereof, wherein each V segment primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complementary to at least one functional a TCR or Ig V region encoding gene segment and wherein said plurality of V segment primers specifically hybridize to substantially all functional TCR or Ig V region encoding gene segments that are present in the composition, and
B) a plurality of J segment oligonucleotide primers that are each independently capable of specifically hybridizing to at least one polynucleotide encoding an TCR or Ig J region polypeptide or to the complement thereof, wherein each J segment primer comprises a nucleotide sequence of at least 15 contiguous nucleotides that is complemen

Assignees

• Adaptive Biotechnologies Corp
• Hutchinson Fred Cancer Res
• Adaptive Biotechnologies Corp

Inventors

• Robins Harlan Saul
• Carlson Christopher Scott
• Rieder Mark J
• Sherwood Anna M
• Emerson Ryan O

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/6881 Primary

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title