# US 11,078,482 B2 · Duplex sequencing using direct repeat molecules

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,078,482 B2
- Document ID: US11078482B2
- Status: Granted
- Kind: B2
- Application number: 16/622,838
- Filed: 2018-06-14
- Published: 2020-06-25
- Granted: 2021-08-03
- Earliest claimed: 2017-06-15
- Assignees: Genome Research Limited
- Applicants: Genome Research Limited
- Inventors: Robert Osborne; Christopher Laumer
- Web page: https://patentgrep.com/patent/US11078482B2

## CPC

- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- 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 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C12Q 1/6888: for detection or identification of organisms
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Described herein is a population of direct repeat molecules, where each molecule of the population contains a direct repeat composed of sequences that are amplified from the opposite strands of a double-stranded fragment of genomic DNA. Within each molecule, the first repeat (referred to as TOP) is amplified from the one strand of a double-stranded fragment of genomic DNA and the second repeat (referred to as BOT′) is amplified from the other strand of the same fragment of double-stranded fragment of genomic DNA.

## Claims

### Claim 1

A population of direct repeat molecules having a strand of the formula:
X-TOP-J-BOT′-Y, wherein:
(i) each direct repeat molecule contains one copy of TOP and one copy of BOT′ and within each repeat molecule TOP and BOT′ are amplified from opposite strands of a double-stranded fragment of genomic DNA and are identical except for positions that correspond to damaged nucleotides in the double-stranded fragment of genomic DNA or errors that occur during amplification;
(ii) TOP and BOT′ vary in the population and have a median length of at least 50 nucleotides;
(iii) X and Y are different, non-complementary sequences of at least 10 nucleotides in length that do not vary in the population; and
(iv) J is a junction sequence.

### Claim 2 (depends on 1)

The population of direct repeat molecules of claim 1, wherein, in the population, TOP and BOT′ each have a complexity of at least 103.

### Claim 3 (depends on 1)

The population of direct repeat molecules of claim 1, wherein the population of direct repeat molecules have a strand of the formula
X-(T)TOP(A)-J-(T)BOT′(A)-Y,
wherein (T) and (A) are thymine and adenine nucleotides that are immediately adjacent to TOP and BOT′.

### Claim 4 (depends on 1)

The population of direct repeat molecules of claim 1, wherein the direct repeat molecules are double-stranded.

### Claim 5 (depends on 1)

The population of direct repeat molecules of claim 1, wherein the genomic DNA is eukaryotic genomic DNA.

### Claim 6 (depends on 5)

The population of direct repeat molecules of claim 5, wherein the genomic DNA is isolated from a tissue biopsy.

### Claim 7 (depends on 5)

The population of direct repeat molecules of claim 5, wherein the genomic DNA is cell-free DNA (cfDNA).

### Claim 8 (depends on 1)

The population of direct repeat molecules of claim 1, wherein the genomic DNA is microbial genomic DNA.

### Claim 9 (depends on 1)

The population of direct repeat molecules of claim 1, wherein the genomic DNA is viral genomic DNA.

### Claim 10 (depends on 1)

The population of direct repeat molecules of claim 1, wherein TOP and BOT′ have a median length of less than 2,000 nucleotides.

### Claim 11 (depends on 1)

The population of direct repeat molecules of claim 1, wherein the molecules are free in solution.

### Claim 12 (depends on 1)

The population of direct repeat molecules of claim 1, wherein J is at least 10 nucleotides in length.

### Claim 13 (depends on 1)

The population of direct repeat molecules of claim 1, wherein J varies in the population.

### Claim 14 (depends on 1)

The population of direct repeat molecules of claim 1, further comprising one or more molecular barcodes.

### Claim 15 (depends on 1)

A method of sequencing, comprising:
(a) producing a population of direct repeat molecules of claim 1 by:
(i) ligating adaptor sequences onto both ends of top and bottom strands of a population of fragments double-stranded genomic DNA to produce double-stranded molecules comprising:
a top strand comprising sequence X at the 5′ end and sequence J at the 3′ end; and
a bottom strand comprising sequence Y′ at the 5′ end, and sequence J′ at the 3′ end; and
(ii) extending the 3′ end of the top strands using the bottom strands as a template, thereby adding the complement of the bottom strands and sequence Y onto the end 3′ end of the top strands;
(b) sequencing the population of direct repeat molecules of claim 1 to obtain the sequences of TOP and BOT′ from the same molecule; and
(c) comparing the sequences of TOP and BOT′ from the same molecule to determine if they are the same or different.

### Claim 16 (depends on 15)

The method of claim 15, further comprising identifying a difference in the sequences of TOP and BOT′ from the same molecule.

### Claim 17 (depends on 16)

The method of claim 16, further comprising excluding the base calls corresponding to a sequence difference from future analysis.

### Claim 18 (depends on 15)

The method of claim 15, wherein the method comprises, prior to sequencing, amplifying the direct repeat molecules.

### Claim 19 (depends on 18)

The method of claim 18, wherein the amplifying is done using primers that hybridize to X and Y, or a complement thereof.

### Claim 20 (depends on 15)

The method of claim 15, wherein the sequencing is done by paired end sequencing.

### Claim 21 (depends on 1)

A method for making a population of direct repeat molecules of claim 1, comprising:
(a) ligating adaptor sequences onto both ends of top and bottom strands of a population of fragments double-stranded genomic DNA to produce double-stranded molecules comprising:
(i) a top strand comprising sequence X at the 5′ end and sequence J at the 3′ end; and
(ii) a bottom strand comprising sequence Y′ at the 5′ end, and sequence J′ at the 3′ end; and
(b) extending the 3′ end of the top strands using the bottom strands as a template, thereby adding the complement of the bottom strands and sequence Y onto the end 3′ end of the top strands.