Immuno-PETE

Patent metadata

Field	Value
Publication number	US-11098360-B2
Application number	US-201715611507-A
Country	US
Kind code	B2
Filing date	Jun 1, 2017
Priority date	Jun 1, 2016
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

Methods and compositions are described herein for primer extension target enrichment of immune receptor (BCR or TCR) sequences.

First claim

What is claimed is:

1. A method for enriching from a sample a plurality of structurally different target polynucleotides, the method comprising:
a) providing a reaction mixture comprising:
i) a plurality of structurally different target polynucleotides, wherein individual target polynucleotides of the plurality comprise immune cell receptor V, J, and optionally C or D gene regions; and
ii) a plurality of immune cell receptor V gene specific primers, wherein the immune cell receptor V gene specific primers comprise at least 10 structurally distinct primers having the following regions from 5′ to 3′: [5′-Phos], [SPLINT], [BARCODE], and [FW], wherein:
[5′-Phos] comprises a 5′ phosphate;
[SPLINT] comprises an adaptor hybridization site of 2-8 nucleotides in length;
[BARCODE] comprises a barcode region of at least 6 nucleotides in length, wherein each nucleotide of the barcode region is independently selected from the group consisting of N and W; and
[FW] of each immune cell receptor V gene specific primer comprises a structurally distinct region that specifically hybridizes to a framework 1, framework 2, or framework 3 region of an immune cell receptor V gene, wherein the immune cell receptor V gene specific primers are hybridized to the V gene regions of the target polynucleotides;

b) extending the hybridized immune cell receptor V gene specific primers with a polymerase, and then removing un-extended immune cell receptor V gene specific primers, if present, wherein the extended immune cell receptor V gene specific primers comprise at least a portion of the immune cell receptor V region, optionally the immune cell receptor D region, at least a portion of the immune cell receptor C region, and at least a portion of the immune cell receptor J region;
c) hybridizing a first universal adaptor to the [SPLINT] adaptor hybridization site of the extended immune cell receptor V gene specific primers; wherein said universal adaptor is a double-stranded adaptor, comprising: a 5′ single-stranded overhang region which can be hybridized to the SPLINT adaptor hybridization site of said extended primers;
d) ligating the hybridized first universal adapters to the extended immune cell receptor V gene specific primers, and then removing un-ligated adapters, if present;
e) hybridizing a plurality of immune cell receptor J gene specific primers to the J region portions of the extended immune cell receptor V gene specific primers, wherein the immune cell receptor J gene specific primers comprise a 3′ J gene hybridizing region and a 5′ second universal adapter region or hybridizing a plurality of immune cell receptor C gene specific primers to the C region portions of the extended immune cell receptor V gene specific primers, wherein the immune cell receptor C gene specific primers comprise a 3′ C gene hybridizing region and a 5′ second universal adapter region; and
f) extending the hybridized immune cell receptor J gene specific primers or C gene specific primers with a polymerase, thereby forming a plurality of structurally different double-stranded products, each comprising at least a portion of the immune cell receptor V region, optionally the immune cell receptor D region, and at least a portion of the immune cell receptor J region or C region flanked by a first and second universal adapter sequence.

2. The method of claim 1 , wherein e) and f) are repeated 2 to 15 times by heating to denature double-stranded products, cooling to hybridize un-extended immune cell receptor J or C gene specific primers to the J region portions of the extended immune cell receptor V gene specific primers or the C region portions of the extended immune cell receptor V gene specific primers, and extending hybridized primers.

3. The method of claim 1 , wherein the removing un-extended immune cell receptor V gene specific primers comprises digesting by single-stranded DNA exonuclease digestion.

4. The method of claim 2 , wherein the method further comprises amplifying double-stranded products comprising first and second universal adapters by universal PCR.

5. The method of claim 2 wherein the method further comprises determining an isotype or clonotype of at least one of the plurality of structurally different double-stranded products or amplified double-stranded products.

6. The method of claim 1 , wherein the [FW] of each immune cell receptor V gene specific primer specifically hybridizes to a framework 1, framework 2, or framework 3 region of a T cell receptor V gene.

7. The method of claim 1 , wherein the [FW] of each immune cell receptor V gene specific primer specifically hybridizes to a framework 1, framework 2, or framework 3 region of a B cell receptor V gene.

8. The method of claim 1 , wherein the plurality comprises at least 10 of the primers set forth in SEQ ID Nos:1-121.

9. The method of claim 1 , wherein the [SPLINT] consists of 6 consecutive nucleotides.

10. The method of claim 1 , wherein the [BARCODE] consists of thirteen consecutive nucleotides selected from the group consisting of N and W.

11. The method of claim 1 , wherein the plurality comprises at least 50 of the primers set forth in SEQ ID NOs:1-121.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Berka Jan
• Godwin Brian
• Dallett Carolina
• Ozturk Sedide
• Platzer Joseph
• Kim Seoyoung

Classifications

• C12Q1/68

  involving nucleic acids · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q1/6881 Primary

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