# US 11,306,356 B2 · Immuno-PETE

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- Number: US 11,306,356 B2
- Document ID: US11306356B2
- Status: Granted
- Kind: B2
- Application number: 16/306,397
- Filed: 2017-06-01
- Published: 2020-12-10
- Granted: 2022-04-19
- Earliest claimed: 2016-06-01
- Assignees: Roche Sequencing Solutions, Inc.
- Applicants: Roche Sequencing Solutions, Inc.
- Inventors: Jan Berka; Brian Godwin; Carolina Dallett; Sedide Ozturk; Joseph Platzer; Seoyoung Kim
- Web page: https://patentgrep.com/patent/US11306356B2

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/68: involving nucleic acids
- 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
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

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

## Claims

### Claim 1

A method for enriching from a sample a plurality of structurally different target polynucleotides, wherein individual target polynucleotides of the plurality comprise immune cell receptor V, J, and C and optionally D gene regions, the method comprising:
a) providing a reaction mixture comprising:
i) the plurality of structurally different target polynucleotides; and
ii) a plurality of immune cell receptor C gene specific primers, wherein the plurality of immune cell receptor C gene specific 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 C gene specific primer comprises a structurally distinct region that specifically hybridizes to an immune cell receptor C gene,
wherein the immune cell receptor C gene specific primers are hybridized to the C gene regions of the target polynucleotides;
b) extending the hybridized immune cell receptor C gene specific primers with a polymerase, and then removing un-extended immune cell receptor C gene specific primers, if present, wherein the extended immune cell receptor C gene specific primers comprise at least a portion of the immune cell receptor C region, optionally the immune cell receptor D region, at least a portion of the immune cell receptor J region, and at least a portion of the immune cell receptor V region;
c) hybridizing a first universal adaptor to the [SPLINT] adaptor hybridization site of the extended immune cell receptor C gene specific primers, wherein said first universal adaptor is a double-stranded adaptor comprising a single-stranded overhang region that hybridizes to the [SPLINT] adaptor hybridization site of said extended primers;
d) ligating the hybridized first universal adapters to the extended immune cell receptor C gene specific primers, and then removing un-ligated adapters, if present;
e) hybridizing a plurality of immune cell receptor V gene specific primers to the V region portions of the extended immune cell receptor C gene specific primers, wherein the immune cell receptor V gene specific primers comprise a 3′ V gene hybridizing region and a 5′ second universal adapter region; and
f) extending the hybridized immune cell receptor V 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 C region flanked by a first and second universal adapter sequence.

### Claim 2 (depends on 1)

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 V gene specific primers to the V region portions of the extended immune cell receptor V gene specific primers, and extending hybridized primers.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 2)

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

### Claim 5 (depends on 4)

The method of claim 4 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.

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

The method of claim 1, wherein the plurality of immune cell receptor C gene specific primers comprises at least 2 of the primers set forth in SEQ ID Nos: 205-213.

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

The method of claim 1, wherein the plurality of immune cell receptor C gene specific primers comprises at least 5 of the primers set forth in SEQ ID NOs: 205-213.