# US 11,441,180 B2 · Compositions and methods for accurately identifying mutations

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- Number: US 11,441,180 B2
- Document ID: US11441180B2
- Status: Granted
- Kind: B2
- Application number: 17/091,676
- Filed: 2020-11-06
- Published: 2021-02-25
- Granted: 2022-09-13
- Earliest claimed: 2012-02-17
- Assignees: Fred Hutchinson Cancer Center
- Applicants: Fred Hutchinson Cancer Research Center
- Inventors: Jason H. Bielas
- Web page: https://patentgrep.com/patent/US11441180B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/81: for yeasts
- C12N 15/85: for animal cells
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6869: Methods for sequencing
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)
- 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)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present disclosure provides compositions and methods for accurately detecting mutations by uniquely tagging double stranded nucleic acid molecules with dual cyphers such that sequence data obtained from a sense strand can be linked to sequence data obtained from an anti-sense strand when sequenced, for example, by massively parallel sequencing methods.

## Claims

### Claim 1

A method of sequencing double-stranded cell-free DNA (cfDNA) molecules, the method comprising:
(a) ligating double-stranded adapters to both ends of the double-stranded cfDNA molecules to form adapter-target complexes, wherein (i) the double-stranded adapters each comprise an identifier sequence, (ii) at least two adapters comprise the same identifier sequence and are ligated to different cfDNA molecules, (iii) cfDNA end sequences together with associated identifier sequences form cyphers at both ends of the cfDNA molecules; and (iv) individual adapter-target complexes comprise different pairs of cyphers;
(b) amplifying the adapter-target complexes to produce a plurality of adapter-target amplification products from each of a first strand and a complementary second strand of the adapter-target complexes;
(c) sequencing the adapter-target amplification products to produce a plurality of first-strand sequencing reads and a plurality of second-strand sequencing reads;
(d) comparing the first-strand sequencing reads with the second-strand sequencing reads for each of a plurality of the adapter-target complexes; and
(e) generating error-corrected sequences for each of a plurality of the double-stranded cfDNA molecules by distinguishing erroneous nucleotides in one strand that lack a matched base change in the complementary strand.

### Claim 2 (depends on 1)

The method of claim 1, wherein the identifier sequence is a random identifier sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein the identifier sequence is not completely random.

### Claim 4 (depends on 1)

The method of claim 1, wherein the identifier sequence is at an end of the double-stranded adapter.

### Claim 5 (depends on 1)

The method of claim 1, wherein the identifier sequence is about 5 to about 50 nucleotides in length.

### Claim 6 (depends on 5)

The method of claim 5, wherein the identifier sequence is 5 to 10 nucleotides in length.

### Claim 7 (depends on 1)

The method of claim 1, wherein the cfDNA end sequences comprise 5 to 20 terminal nucleotides of the double-stranded cfDNA molecules.

### Claim 8 (depends on 1)

The method of claim 1, wherein the erroneous nucleotides comprise a polymerase error that arose during amplification or sequencing.

### Claim 9 (depends on 1)

The method of claim 1, further comprising identifying a true mutation that is present in both strands of one of the double-stranded cfDNA molecules.

### Claim 10 (depends on 9)

The method of claim 9, wherein the true mutation is identified when it is present in substantially all first-strand sequencing reads and second-strand sequencing reads for the double-stranded cfDNA molecule.

### Claim 11 (depends on 10)

The method of claim 10, wherein the true mutation is identified when it is present in all first-strand sequencing reads and second-strand sequencing reads for the double-stranded cfDNA molecule.

### Claim 12 (depends on 1)

The method of claim 1, wherein generating error-corrected sequences comprises comparing the first-strand sequencing reads with the second-strand sequencing reads within each of a plurality of groups of reads, wherein the groups of reads are distinguishable by the different pairs of cyphers.

### Claim 13 (depends on 1)

The method of claim 1, wherein the cfDNA molecules are isolated from a blood sample of a subject.

### Claim 14 (depends on 1)

The method of claim 1, wherein the cfDNA molecules are isolated from a subject having cancer.

### Claim 15 (depends on 1)

The method of claim 1, further comprising detecting a cfDNA molecule from a cancer.