# US 11,898,204 B2 · Generation of single-stranded circular DNA templates for single molecule sequencing

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- Number: US 11,898,204 B2
- Document ID: US11898204B2
- Status: Granted
- Kind: B2
- Application number: 16/977,421
- Filed: 2019-02-28
- Published: 2023-06-15
- Granted: 2024-02-13
- Earliest claimed: 2018-03-02
- Assignees: Roche Sequencing Solutions, Inc.; Kapa Biosystems, Inc.
- Applicants: Roche Sequencing Solutions, Inc.; Kapa Biosystems, Inc.
- Inventors: Aruna Arcot; Daleen Badenhorst; Rui Chen; Toummy Guettouche; Ashley Hayes; Jenny A. Johnson; Severine Margeridon; Martin Ranik; Persis Wadia; Alexandra Hui Wang
- Web page: https://patentgrep.com/patent/US11898204B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6855: Ligating adaptors
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention is a novel method of separately sequencing each strand of a nucleic acid involving the use of an adaptor comprising a strand cleavage site or a strand synthesis termination site. The adaptor may also be self-priming at the strand cleavage site.

## Claims

### Claim 1

A method of separately sequencing each strand of a target nucleic acid comprising the steps of:
a) in a reaction mixture, joining the ends of a double stranded target nucleic acid to adaptors to form a doubly-adapted target nucleic acid wherein the adaptor is a single strand forming a double stranded stem and a single-stranded loop and the stem comprises at least one strand cleavage site;
b) contacting the reaction mixture with an exonuclease thereby enriching for the doubly-adapted target nucleic acid;
c) contacting the reaction mixture with a cleavage agent to cleave the doubly-adapted target nucleic acid at the cleavage sites forming extendable termini on each strand;
d) extending the extendable termini thereby separately sequencing each strand of the target nucleic acid, wherein extension is terminated at the cleavage site and does not proceed onto the complementary strand.

### Claim 2 (depends on 1)

The method of claim 1, wherein joining to the adaptor is by ligation.

### Claim 3 (depends on 1)

The method of claim 1, wherein the adaptor comprises at least one barcode.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cleavage site comprises one or more deoxyuracils and the cleavage agent comprises Uracil-DNA-N-glycosylase (UNG) and an endonuclease.

### Claim 5 (depends on 1)

The method of claim 1, wherein the adaptor comprises an exonuclease protection nucleotide.

### Claim 6 (depends on 1)

The method of claim 1, wherein the adaptor comprises a ligand for a capture moiety.

### Claim 7 (depends on 1)

The method of claim 1, further comprising a target enrichment step prior to sequencing.

### Claim 8 (depends on 7)

The method of claim 7 wherein the enrichment is by capture via target-specific probes.

### Claim 9

A method of determining the sequence of a library of a target nucleic acids for separately sequencing each strand of a target nucleic acid, the method comprising the steps of:
a) in a reaction mixture, joining the ends of double stranded target nucleic acids to adaptors to form a doubly-adapted target nucleic acids wherein the adaptor is a single strand forming a double stranded stem and a single-stranded loop and the stem comprises a strand cleavage site;
b) contacting the reaction mixture with an exonuclease thereby enriching for the doubly-adapted target nucleic acids;
c) contacting the reaction mixture with a cleavage agent to cleave the doubly-adapted target nucleic acids at the cleavage sites forming extendable termini on each strand;
d) extending the extendable termini thereby separately sequencing each strand of the target nucleic acids in the library, wherein the extension is terminated at the cleavage site and does not proceed onto the complementary strand.

### Claim 10

A method of separately sequencing each strand of a target nucleic acid comprising the steps of:
a) in a reaction mixture, joining the ends of a double stranded target nucleic acid to adaptors to form a doubly-adapted target nucleic acid wherein the adaptor is a single strand forming a double stranded stem and a single-stranded loop, and the loop comprises a primer binding site and the adaptor further comprises strand synthesis termination site selected from an abasic nucleotide and a non-nucleotide linker and;
b) contacting the reaction mixture with an exonuclease thereby enriching for the doubly-adapted target nucleic acid;
c) contacting the reaction mixture with a primer capable of hybridizing to the primer binding sites;
d) extending the primers thereby separately sequencing each strand of the target nucleic acid, wherein extension is terminated at the strand synthesis termination site and does not proceed onto the complementary strand.

### Claim 11

A method of determining the sequence of a library for separately sequencing each strand of a target nucleic acid comprising the steps of:
a) in a reaction mixture, joining the ends of double stranded target nucleic acids to adaptors to form doubly-adapted target nucleic acids wherein the adaptor is a single strand forming a double stranded stem and a single-stranded loop, and the loop comprises a primer binding site and the adaptor further comprises strand synthesis termination site;
b) contacting the reaction mixture with an exonuclease thereby enriching for the doubly-adapted target nucleic acids and forming a library of doubly-adapted target nucleic acids;
c) contacting the library with a primer capable of hybridizing to the primer binding sites;
d) extending the primers thereby separately sequencing each strand of the target nucleic acids in the library, wherein extension is terminated at the strand synthesis termination site and does not proceed onto the complementary strand.

### Claim 12 (depends on 1)

The method of claim 1, wherein the cleavage site is an abasic site and the cleavage agent is AP nuclease.

### Claim 13 (depends on 1)

The method of claim 1, wherein the cleavage site is an 8-oxoguanine and the cleavage agent is 8-oxoguanine DNA glycosylase.

### Claim 14 (depends on 1)

The method of claim 1, wherein the cleavage site is a deoxyinosine and the cleavage agent is a combination of alkyladenine glycosylase (AAG) and endonuclease.

### Claim 15 (depends on 1)

The method of claim 1, wherein the cleavage site is a ribonucleotide and the cleavage agent is RNaseH.