Granted Patent B2

US 12,747,473 · App. 17/219,543 · Granted Sep 29, 2026

Inventor: Jason H. Bielas (Seattle, WA)

Assignee: Fred Hutchinson Cancer Center

C12Q1/6874C12N15/10C12N15/1065C12N15/1093C12N15/70C12N15/81C12N15/85C12Q1/6827C12Q1/6869C40B40/08C40B50/06

Abstract

Claims (31)

1 . A method of assaying sample nucleic acid molecules from a subject for a target mutation, the method comprising:

(a) joining the sample nucleic acid molecules to cypher polynucleotides to form double-stranded cypher-target nucleic acid complexes, wherein:

(i) the joining comprises ligation;

(ii) each cypher polynucleotide comprises a bar code sequence selected from a plurality of distinct bar code sequences; and

(iii) bar code sequences alone or in combination with sample nucleic acid molecule end sequences distinguish cypher-target nucleic acid complexes from one another;

(b) amplifying the cypher-target nucleic acid complexes to produce a plurality of cypher-target amplification products from first strands and complementary second strands of the cypher-target nucleic acid complexes;

(c) sequencing the cypher-target amplification products to produce a plurality of first-strand sequencing reads and a plurality of second-strand sequencing reads;

(d) grouping sequencing reads based on (i) the bar code sequences and (ii) the nucleic acid molecule sequences, wherein a group comprises first-strand sequencing reads and second-strand sequencing reads from the cypher-target amplification products of one of the cypher-target nucleic acid complexes;

(e) generating error-corrected sequences of the sample nucleic acid molecules by comparing the first-strand sequencing reads and second-strand sequencing reads within groups of sequencing reads to distinguish erroneous nucleotides in one strand that lack a matched base change in the complementary strand; and

(f) determining whether the target mutation is present in one or more of the sample nucleic acid molecules by comparing the error-corrected sequences to a reference sequence comprising the target mutation.

2 . The method of claim 1 , wherein the sample nucleic acid molecules comprise a mutation present at a frequency of 2.1×10 −6 or lower.

3 . The method of claim 1 , wherein generating the error-corrected sequences results in a measureable sequencing error rate from about 10 −6 to about 10 −8 .

4 . The method of claim 1 , wherein the plurality of first-strand sequencing reads and the plurality of second-strand sequencing reads are filtered based on assigned quality scores.

5 . The method of claim 1 , wherein the subject was previously diagnosed with a cancer.

6 . The method of claim 1 , wherein the sample nucleic acid molecules are obtained from a blood sample of the subject.

7 . The method of claim 1 , wherein the sample nucleic acid molecules are obtained from plasma of the subject.

8 . The method of claim 1 , wherein the sample nucleic acid molecules are derived from cancer cells of the subject.

9 . The method of claim 1 , wherein the ligation comprises ligating to an overhang or a blunt end.

10 . The method of claim 1 , wherein the cypher polynucleotides comprising the bar code sequences are contained within a pool of cypher polynucleotides comprising known sequences.

11 . The method of claim 1 , wherein the joining comprises treating the sample nucleic acid molecules to blunt the ends thereof prior to the ligation.

12 . The method of claim 1 , further comprising purifying a plurality of cypher-target nucleic acid complexes prior to sequencing, wherein the purified cypher-target nucleic acid complexes comprise nucleic acid molecules from specific genomic regions.

13 . The method of claim 1 , further comprising purifying a plurality of cypher-target nucleic acid complexes prior to sequencing, wherein the purified cypher-target nucleic acid complexes comprise one or more target sequences.

14 . The method of claim 1 , wherein grouping sequencing reads is based on (i) the bar code sequences and (ii) end sequences of the nucleic acid molecule.

15 . The method of claim 1 , wherein the ligation comprises ligating cypher polynucleotides to both ends of the sample nucleic acid molecules.

16 . The method of claim 14 , wherein the bar code sequences at both ends together form a unique pair of identifiers that differ between each of the other pairs of identifiers ligated to the sample nucleic acid molecules.

17 . The method of claim 1 , wherein the reference sequence is a human genomic sequence.

18 . The method of claim 1 , further comprising measuring a frequency of sample nucleic acid molecules comprising the target mutation.

19 . The method of claim 1 , wherein the error-corrected sequences comprise only nucleotide bases at which the first-strand sequencing reads and second-strand sequencing reads are in agreement.

20 . The method of claim 1 , wherein the target mutation is a transition mutation, a somatic mutation, or a mitochondrial mutation.

21 . The method of claim 1 , wherein the bar code sequences comprise random or partially random sequences.

22 . The method of claim 1 , wherein the bar code sequences comprise nonrandom sequences.

Assignments (2)

Continuity (7)

References Cited (216)