Compositions and methods for immune repertoire sequencing

Patent metadata

Field	Value
Publication number	US-11970787-B2
Application number	US-202117248722-A
Country	US
Kind code	B2
Filing date	Feb 4, 2021
Priority date	Jan 17, 2017
Publication date	Apr 30, 2024
Grant date	Apr 30, 2024

Abstract

The present disclosure provides methods, compositions, kits, and systems useful in the determination and evaluation of the immune repertoire. In one aspect, target-specific primer panels provide for the effective amplification of sequences of T cell receptor and/or B cell receptor chains with improved sequencing accuracy and resolution over the repertoire. Variable regions associated with the immune cell receptor are resolved to effectively portray clonal diversity of a biological sample and/or differences associated with the immune cell repertoire of a biological sample.

First claim

What is claimed is:

1. A method for preparing an immune receptor repertoire library, comprising:
A. generating target immune receptor amplicon molecules comprising performing a single multiplex amplification reaction to amplify expressed target immune receptor nucleic acid template molecules using at least one set of:
i) (a) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 1 (FRI) within the V gene,
(b) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 2 (FR2) within the V gene, or
(c) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene; and
ii) (a) one or more C gene primers directed to at least a portion of a C gene of the at least one immune receptor coding sequence, or
(b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the at least one immune receptor coding sequence;
wherein each set of i) and ii) primers is directed to coding sequences of the same target immune receptor gene selected from a T cell receptor gene or an antibody receptor gene and wherein performing the amplification using the at least one set of i) and ii) primers results in amplicon molecules representing a target immune receptor repertoire in a sample, thereby generating immune receptor amplicon molecules;
B. treating the amplicon molecules to form blunt-ended amplicon molecules; and
C. ligating at least one adapter to at least one of the treated amplicon molecules, thereby producing a library of adapter-ligated target immune receptor amplicon molecules comprising the target immune receptor repertoire;
wherein each of the plurality of V gene primers, the plurality of J gene primers and/or the one of more C gene primers includes two or more modified cleavable nucleotides within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence.

2. The method of claim 1 , wherein each of the plurality of V gene primers, the plurality of J gene primers and/or the one of more C gene primers has any one or more of the following criteria:
(1) length is about 15 to about 40 bases in length;
(2) Tm of from above 60° C. to about 70° C.;
(3) has low cross-reactivity with non-target sequences present in the sample;
(4) at least the first four nucleotides (going from 3′ to 5′ direction) are non-complementary to any sequence within any other primer present in the same reaction; and
(5) are non-complementary to any consecutive stretch of at least 5 nucleotides within any other produced target amplicon.

3. The method of claim 1 , wherein the at least one set of i) and ii) is i)(a) and ii)(a), wherein the plurality of V gene primers anneal to at least a portion of the FRI region of the template molecules, and wherein the one or more C gene primers comprises at least two primers that anneal to at least a portion of the C gene portion of the template molecules.

4. The method of claim 3 , wherein the generated immune receptor amplicons are about 300 to about 600 nucleotides in length.

5. The method of claim 3 , wherein the at least one set of i) and ii) is selected from the primers of SEQ ID NO: 1-180 and SEQ ID NO: 181-184.

6. The method of claim 3 , wherein the plurality of V gene primers is about 45 to about 90 different V gene primers.

7. The method of claim 1 , wherein the at least one set of i) and ii) is i)(a) and ii)(b), wherein the plurality of V gene primers anneal to at least a portion of the FRI region of the template molecules, and wherein the plurality of J gene primers comprises at least ten primers that anneal to at least a portion of the J gene portion of the template molecules.

8. The method of claim 7 , wherein the at least one set of i) and ii) is selected from the primers of Table 2 and Table 5.

9. The method of claim 1 , wherein the at least one set of i) and ii) is i)(c) and ii)(a), wherein the plurality of V gene primers anneal to at least a portion of the FR3 region of the template molecules, and wherein the one or more C gene primers comprises at least two primers that anneal to at least a portion of the C gene portion of the template molecules.

10. The method of claim 9 , wherein the at least one set of i) and ii) is selected from the primers of Table 3 and Table 4.

11. The method of claim 1 , wherein the at least one set of i) and ii) is i)(c) and ii)(b), wherein the plurality of V gene primers anneal to at least a portion of the FR3 region of the template molecules, and wherein the plurality of J gene primers comprises at least ten primers that anneal to at least a portion of the J gene portion of the template molecules.

12. The method of claim 11 , wherein the at least one set of i) and ii) is selected from the primers of Table 3 and Table 5.

Assignees

• Life Technologies Corp

Inventors

• Looney Timothy
• Lowman Geoffrey
• Lin Lifeng

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C40B50/06 Primary

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title