METHODS, COMPOSITIONS AND SYSTEMS FOR CALIBRATING EPIGENETIC PARTITIONING ASSAYS

• US 20200131566A1
• Filed: 10/31/2019
• Published: 04/30/2020
• Est. Priority Date: 10/31/2018
• Status: Active Application

• Current Assignee: Guardant Health Inc.
• Original Assignee: Guardant Health Inc.
• Inventors: KENNEDY, Andrew, WESTESSON, Oscar, He, Yupeng, SCHULTZ, Matthew
• Application Number:
  US16/670,827
• Publication Number:
  US 20200131566A1
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation

Abstract

In an aspect, a method for evaluating the partitioning of nucleic acid molecules in a sample of polynucleotides based on epigenetic state, comprising: (a) adding a set of epigenetic-control nucleic acid molecules to the nucleic acid molecules in the sample of polynucleotides, whereby producing a spiked-in sample; (b) partitioning nucleic acid molecules of the spiked-in sample into plurality of partitioned sets; (c) enriching a subset of molecules from the plurality of partitioned sets to generate enriched molecules, wherein the enriched molecules comprises a group of epigenetic-control nucleic acid molecules and a group of nucleic acid molecules from the sample of polynucleotides; (d) sequencing the enriched molecules to produce sequencing reads; (e) analyzing the sequencing reads to generate one or more epigenetic partition scores of the epigenetic-control nucleic acid molecules; and (f) comparing the one or more epigenetic partition scores with one or more epigenetic partition cut-offs.

Claims

1-38. -38. (canceled)

39. A method for evaluating partitioning of nucleic acid molecules in a sample of polynucleotides based on epigenetic state, comprising:

a. adding a set of epigenetic-control nucleic acid molecules to the nucleic acid molecules in the sample of polynucleotides, thereby producing a spiked-in sample;

b. partitioning nucleic acid molecules of at least a subset of the spiked-in sample into a plurality of partitioned sets;

c. enriching at least a subset of molecules from the plurality of partitioned sets to generate a set of enriched molecules, wherein the set of enriched molecules comprises a group of epigenetic-control nucleic acid molecules and a group of nucleic acid molecules from the sample of polynucleotides;

d. sequencing at least a subset of the set of enriched molecules to produce a set of sequencing reads;

e. analyzing at least a subset of the set of sequencing reads to generate one or more epigenetic partition scores of the epigenetic-control nucleic acid molecules; and

f. comparing the one or more epigenetic partition scores with one or more epigenetic partition cut-offs.

42. The method of claim 39, further comprising tagging the nucleic acid molecules in a partitioned set of the plurality of partitioned sets with a set of tags to produce a population of tagged nucleic acid molecules, wherein the tagged nucleic acid molecules comprise one or more tags.

43. The method of claim 42, wherein the set of tags used in a first partitioned set of the plurality of partitioned sets is different from the set of tags used in a second partitioned set of the plurality of partitioned sets.

44. The method of claim 43, wherein the set of tags are attached to the nucleic acid molecules by ligation of adapters to the nucleic acid molecules, wherein the adapters comprise one or more tags.

45. The method of claim 39, further comprising g) classifying the method as (i) being successful, if the one or more epigenetic partition scores of the epigenetic-control nucleic acid molecules;

or (ii) being unsuccessful, if at least one of the one or more epigenetic partition scores of the epigenetic control molecules.

46. The method of claim 39, wherein the set of epigenetic-control nucleic acid molecules comprises two or more subsets of epigenetic-control nucleic acid molecules, wherein a subset of the two or more subsets of epigenetic-control nucleic acid molecules comprises a plurality of epigenetic-control nucleic acid molecules comprising an epigenetic modification region.

47. The method of claim 46, wherein the epigenetic-control nucleic acid molecule further comprises an identifier region.

48. The method of claim 47, wherein the identifier region is on one or both sides of the epigenetic modification region of the epigenetic-control nucleic acid molecules.

49. The method of claim 46, wherein the epigenetic modification region of the epigenetic-control nucleic acid molecules in at least one subset comprises at least one nucleotide with epigenetic modification.

50. The method of claim 49, wherein the subset comprises epigenetic-control nucleic acid molecules with a same number of nucleotides with epigenetic modification.

51. The method of claim 49, wherein the number of nucleotides with epigenetic modification in a first subset is different from the number of nucleotides with epigenetic modification in a second subset.

52. The method of claim 47, wherein the identifier region of the epigenetic-control nucleic acid molecules comprises a molecular barcode.

54. The method of claim 47, wherein the identifier region further comprises at least one epigenetic state barcode.

55. The method of claim 47, wherein the identifier region comprises one or more primer binding sites.

57. The method of claim 49, wherein the epigenetic modification is DNA methylation.

58. The method of claim 49, wherein the nucleotide with epigenetic modification comprises a methylated nucleotide.

59. The method of claim 58, wherein the methylated nucleotide comprises 5-methylcytosine.

61. The method of claim 46, wherein each subset of epigenetic-control nucleic acid molecules is in equimolar concentration.

62. The method of claim 46, wherein each subset of epigenetic-control nucleic acid molecules is in non-equimolar concentration.

63. The method of claim 58, wherein the number of methylated nucleotides in the epigenetic-control nucleic acid molecules in at least one of the subsets is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40 or at least 50.

65. The method of claim 39, wherein the epigenetic state is a methylation level of the nucleic acid molecules.

66. The method of claim 57, wherein the plurality of partitioned sets comprises nucleic acid molecules of the spiked-in sample partitioned based on the methylation level of the nucleic acid molecules.

67. The method of claim 46, wherein the epigenetic modification region of the epigenetic-control nucleic acid molecules comprises of a length of about 160 bp.

68. The method of claim 39, wherein the sequencing of the plurality of enriched molecules is performed by a nucleic acid sequencer.

70. The method of claim 46, wherein the epigenetic modification region of the epigenetic-control nucleic acid molecules comprises a nucleic acid sequence corresponding to a non-human genome.

72. The method of claim 39, wherein the nucleic acid molecules in the sample of polynucleotides are cell-free deoxyribonucleic acid (cfDNA) molecules.

73. The method of claim 72, wherein the number of methylated nucleotides in the epigenetic-control nucleic acid molecule in at least one of the subsets is 0, 2, 4, 6, 8, 10, 12, 14, at least 16, at least 20, at least 30, at least 40 or at least 50.

76. The method of claim 39, wherein the partitioning comprises partitioning the nucleic acid molecules based on a differential binding affinity of the nucleic acid molecules to a binding agent that preferentially binds to nucleic acid molecules comprising nucleotides with epigenetic modification.

40-41. -41. (canceled)

53. (canceled)

56. (canceled)

60. (canceled)

64. (canceled)

69. (canceled)

71. (canceled)

74-75. -75. (canceled)

77-95. -95. (canceled)