METHODS AND SYSTEMS FOR DETECTING GENETIC VARIANTS

• US 20200131568A1
• Filed: 12/13/2019
• Published: 04/30/2020
• Est. Priority Date: 12/28/2013
• Status: Active Grant

• Current Assignee: Guardant Health Inc.
• Original Assignee: Guardant Health Inc.
• Inventors: TALASAZ, AmirAli, MORTIMER, Stefanie Ann Ward
• Granted Patent: US 10,889,858 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Abstract

Disclosed herein in are methods and systems for determining genetic variants (e.g., copy number variation) in a polynucleotide sample. A method for determining copy number variations includes tagging double-stranded polynucleotides with duplex tags, sequencing polynucleotides from the sample and estimating total number of polynucleotides mapping to selected genetic loci. The estimate of total number of polynucleotides can involve estimating the number of double-stranded polynucleotides in the original sample for which no sequence reads are generated. This number can be generated using the number of polynucleotides for which reads for both complementary strands are detected and reads for which only one of the two complementary strands is detected.

Claims

1-30. -30. (canceled)

31. A method for processing polynucleotide molecules from a sample of a subject, comprising:

exposing the polynucleotide molecules from the sample to a set of library adaptors comprising a plurality of molecular barcodes to generate tagged parent polynucleotides,

wherein exposing comprises ligating a plurality of library adaptors from the set of library adaptors to some of the polynucleotide molecules form the sample using at least a 10×

molar excess of library adaptors as compared to the polynucleotide molecules;

wherein exposing yields at least 20% of the polynucleotide molecules from the sample having library adaptors ligated to both ends of a molecule of the polynucleotide molecules.

32. The method of claim 31, wherein the polynucleotide molecules are cell-free deoxyribonucleic acid (cfDNA) molecules.

33. The method of claim 31, wherein the polynucleotide molecules are fragmented nucleic acid molecules.

34. The method of claim 31, wherein the sample is selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.

35. The method of claim 31, wherein the library adaptors are ligated to the polynucleotide molecules by blunt-end ligation or sticky-end ligation.

36. The method of claim 31, wherein the ligating comprises using more than a 100×

molar excess of library adaptors as compared to the polynucleotide molecules.

37. The method of claim 31, wherein at least 40% of the polynucleotide molecules from the sample are attached to library adaptors at both ends of a molecule of the polynucleotide molecules.

38. The method of claim 31, wherein the molecular barcodes in the plurality of library adaptors have 2 to 100,000 different molecular barcode sequences.

39. The method of claim 31, wherein the molecular barcodes in the plurality of library adaptors have 5 to 100 different molecular barcode sequences.

40. The method of claim 39, wherein the molecular barcodes have a length of 5 to 20 nucleotides.

41. The method of claim 31, wherein the molecular barcodes are different from one another and have an edit distance of at least 1 between one another.

42. The method of claim 31, wherein the library adaptors are Y-shaped adaptors.

43. The method of claim 31, further comprising amplifying a plurality of the tagged parent polynucleotides to generate amplified progeny polynucleotides.

44. The method of claim 43, further comprising selectively enriching the amplified progeny polynucleotides for a plurality of genomic regions of interest.

45. The method of claim 44, wherein the plurality of genomic regions of interest comprises exon sequences.

46. The method of claim 44, the wherein the genomic regions of interest comprise sequences from one or more genes selected from the group consisting of:

ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RB1, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSF1R, CTNNB1, ERBB4, EZH2, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA, and NTRK1.

47. The method of claim 44, wherein selectively enriching the subset of amplified progeny polynucleotides for a plurality of genomic regions of interest comprises using a set of probes that hybridize to the plurality of genomic regions of interest.

48. The method of claim 44, further comprising amplifying a subset of enriched amplified progeny polynucleotides.

49. A method for analyzing polynucleotide molecules from a sample of a subject, comprising:

(a) exposing the polynucleotide molecules from the sample to a set of library adaptors comprising a plurality of molecular barcodes to generate tagged parent polynucleotides,

wherein exposing comprises ligating a plurality of library adaptors from the set of library adaptors to some of the polynucleotide molecules form the sample using at least a 10×

molar excess of library adaptors as compared to the polynucleotide molecules;

wherein exposing yields at least 20% of the polynucleotide molecules from the sample having library adaptors ligated to both ends of a molecule of the polynucleotide molecules;

(b) subjecting a plurality of the tagged parent polynucleotides to amplification reactions under conditions that yield amplified progeny polynucleotides;

(c) selectively enriching the amplified progeny polynucleotides for genomic regions of interests;

(d) determining nucleotide sequences of a plurality of enriched amplified progeny polynucleotides; and

(e) analyzing the nucleotide sequences with a programed computer processor to identify one or more genetic variants in the sample of the subject.

50. The method of claim 49, wherein the polynucleotide molecules are cell-free deoxyribonucleic acid (cfDNA) molecules.

51. The method of claim 49, wherein the polynucleotide molecules are fragmented nucleic acid molecules.

52. The method of claim 49, wherein the sample is selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.

53. The method of claim 49, wherein the library adaptors are ligated to the polynucleotide molecules by blunt-end ligation or sticky-end ligation.

54. The method of claim 49, wherein the ligating comprises using more than a 100×

molar excess of library adaptors as compared to the polynucleotide molecules.

55. The method of claim 49, wherein at least 40% of the polynucleotide molecules from the sample are attached to library adaptors at both ends of a molecule of the polynucleotide molecules.

56. The method of claim 49, wherein the genomic regions of interest comprise sequences from one or more genes selected from the group consisting of:

ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RB1, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSF1R, CTNNB1, ERBB4, EZH2, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA, and NTRK1.

57. The method of claim 49, wherein the analyzing in (e) further comprises mapping a plurality of the nucleotide sequences to a reference sequence.

58. The method of claim 57, further comprising sorting the nucleotide sequences into families based on a combination of the molecular barcode sequences and the mapping positions of the nucleotide sequences that map to a reference sequence, wherein a family is representative of a tagged parent polynucleotide.

59. The method of claim 58, further comprising determining a consensus sequence for each of the families, and identifying consensus sequences having a genetic variant as compared to the reference sequence.

60. The method of claim 49, wherein the one or more genetic variants comprise a cancer-associated somatic mutation.