Compositions and methods for accurately identifying mutations

Patent metadata

Field	Value
Publication number	US-2024417790-A1
Application number	US-202418620758-A
Country	US
Kind code	A1
Filing date	Mar 28, 2024
Priority date	Feb 17, 2012
Publication date	Dec 19, 2024
Grant date	—

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.

First claim

1 .- 38 . (canceled)

39 . A method for sequencing double-stranded target nucleic acid molecules, comprising:
(a) ligating the double-stranded target nucleic acid molecules to double-stranded cyphers at both ends to form cypher-target nucleic acid complexes, wherein the double-stranded cyphers each comprise a random or partially random identifier sequence, and further wherein each target nucleic acid molecule is a DNA molecule comprising a target tag sequence that together with the double-stranded cyphers uniquely labels the double-stranded target nucleic acid molecule; (b) amplifying each strand of the cypher-target nucleic acid complexes to produce a plurality of cypher-target amplification products from each of a first strand and a complementary second strand 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, wherein the plurality of first-strand sequencing reads and the plurality of second-strand sequencing reads each comprise an identifier sequence and a target tag sequence; (d) grouping the first-strand sequencing reads and second-strand sequencing reads based on sequence identity of: the identifier sequence, and the target tag sequence, wherein a group comprises sequencing reads from one of the uniquely labeled double-stranded target nucleic acid molecules; and (e) comparing the first-strand sequencing reads with the second-strand sequencing reads in each group, and generating an error-corrected sequence for a plurality of the double-stranded target nucleic acid molecules by distinguishing erroneous nucleotides in one strand that lack a matched base change in the complementary strand.

40 . The method of claim 39 , wherein the double-stranded cyphers comprise random identifier sequences.

41 . The method of claim 39 , wherein the double-stranded cyphers comprise identifier sequences that are not completely random.

42 . The method of claim 39 , wherein a target tag sequence comprises nucleotides at an end of a target nucleic acid molecule.

43 . The method of claim 42 , wherein the end of the target nucleic acid molecule is a sheared end.

44 . The method of claim 39 , wherein the random or partially-random identifier sequence is double-stranded.

45 . The method of claim 39 , wherein the identifier sequence is at an end of the double-stranded cypher.

46 . The method of claim 39 , wherein the target tag sequence is 5 nucleotides to 20 nucleotides in length.

47 . The method of claim 39 , wherein each of the cyphers are unique.

48 . The method of claim 39 , wherein the erroneous nucleotides comprise a polymerase error that arose during amplification or sequencing.

49 . The method of claim 39 , wherein the plurality of target nucleic acid molecules comprise a mutation present at a frequency of 2.1×10 −6 or lower.

50 . The method of claim 39 , further comprising detecting a cancer biomarker in one of the error-corrected sequences, wherein the cancer biomarker comprises a nucleotide mutation.

51 . The method of claim 39 , further comprising using the error-corrected sequences to assess cancer response to therapy.

52 . The method of claim 39 , wherein the sample is derived from a human subject having cancer, and wherein the method further comprises detecting in one of the error-corrected sequences a mutation that confers to the cancer resistance to cancer therapy.

Assignees

• Fred Hutchinson Cancer Center

Inventors

• Bielas Jason H

Classifications

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N15/81

  for yeasts · CPC title