# US 12,110,534 B2 · Generation of single-stranded circular DNA templates for single molecule sequencing

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- Number: US 12,110,534 B2
- Document ID: US12110534B2
- Status: Granted
- Kind: B2
- Application number: 16/945,099
- Filed: 2020-07-31
- Published: 2021-04-22
- Granted: 2024-10-08
- Earliest claimed: 2018-02-05
- Assignees: Roche Sequencing Solutions, Inc.; Kapa Biosystems, Inc.
- Applicants: Roche Sequencing Solutions, Inc.; Kapa Biosystems, Inc.
- Inventors: Daleen Badenhorst; Michael S. Berry; Richard Dannebaum; Ashley Hayes; Severine Margeridon; Martin Ranik; Etienne Slabbert
- Web page: https://patentgrep.com/patent/US12110534B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q 2521/319: Exonuclease
- C12Q 2525/155: incorporating/generating a new priming site
- C12Q 2525/191: incorporating an adaptor
- C12Q 2525/307: Circular oligonucleotides
- C12Q 2535/122: Massive parallel sequencing
- 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

## Abstract

The invention is a novel method of sequencing nucleic acids involving making and sequencing a library of single stranded circular target nucleic acids.

## Claims

### Claim 1

A method of separately sequencing each strand of a double-stranded target nucleic acid, wherein the method comprises the following steps:
(a) in a reaction mixture, joining a double-stranded target nucleic acid to an adaptor to form an adapted target nucleic acid, wherein the adaptor comprises primer-binding sites, and wherein the adaptor does not consist of a long strand and a short strand;
(b) amplifying the adapted target nucleic acid with a pair of primers, wherein the pair of primers are complementary to the primer-binding sites, thereby forming an amplicon, wherein one primer in the pair of primers comprises a modified nucleotide affecting a rate of digestion by an exonuclease;
(c) contacting the reaction mixture with an exonuclease, thereby eliminating from the reaction mixture the first of the two complementary strands of the amplicon;
(d) circularizing the second of the two complementary strands of the amplicon to form a single stranded circle;
(e) annealing a sequencing primer to the single-stranded circle; and
(f) extending the primer, thereby sequencing one strand of the target nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the joining of the double-stranded target nucleic acid to the adaptor is by ligation.

### Claim 3 (depends on 2)

The method of claim 2, wherein the ligation is by joining of cohesive ends of the target nucleic acid and the adaptor.

### Claim 4 (depends on 1)

The method of claim 1, wherein the adaptor comprises a double-stranded part and a single-stranded part comprising two non-annealed portions.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

The method of claim 1, wherein the modified nucleotide is a 5′-phosphorylated terminal nucleotide and the exonuclease is Lambda exonuclease.

### Claim 7 (depends on 1)

The method of claim 1, further comprising a second exonuclease digestion step, after the circularization step (d).

### Claim 8 (depends on 1)

The method of claim 1, wherein circularization is by splint ligation.

### Claim 9 (depends on 1)

The method of claim 1, wherein the sequencing primer-binding site is in the adaptor.

### Claim 10 (depends on 1)

The method of claim 1, further comprising, prior to step (c), contacting the reaction mixture with a DNA damage-specific agent selected from glycosylase and endonuclease.

### Claim 11 (depends on 1)

The method of claim 1, wherein the steps (a)-(f) are performed on a plurality of target nucleic acids in a sample thereby forming and sequencing a library of nucleic acids from the sample.