Granted Patent B2

US 12,735,751 · App. 19/362,169 · Granted Sep 15, 2026

Inventor: Amirali Talasaz (Atherton, CA)

Assignee: GUARDANT HEALTH, INC.

C12Q1/6886C12N15/1065C12Q1/6806C12Q1/6869C12Q1/6874G16B30/00G16B30/10C12Q2600/118C12Q2600/156C12Q2600/158C12Q2600/16

Abstract

Claims (25)

1 . A method for detecting methylation profiles in cell-free nucleic acid molecules from a sample from a subject, comprising:

(a) attaching adapters to a plurality of the cell-free nucleic acid molecules to generate parent polynucleotides;

(b) amplifying a plurality of the parent polynucleotides to produce a corresponding set of amplified progeny polynucleotides;

(c) sequencing at least a subset of the set of amplified progeny polynucleotides to produce a set of sequencing reads; and

(d) collapsing and analyzing the set of sequencing reads, which comprises: (i) grouping sequencing reads sequenced from amplified progeny polynucleotides into families, each family amplified from the same parent polynucleotide; (ii) the analysis of polymorphic forms including methylation, hypermethylation, and/or hydroxymethylation; and (iii) using one or more bioinformatics processes to detect methylation profiles.

2 . The method of claim 1 , wherein the sample is selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.

3 . The method of claim 1 , wherein the sample comprises between 1 nanogram (ng) and 100 ng of the cell-free nucleic acid molecules.

4 . The method of claim 1 , wherein the plurality of cell-free nucleic acid molecules comprises 100 to 100,000 haploid human genome equivalents of cell-free nucleic acid.

5 . The method of claim 1 , wherein the attaching comprises tagging by enzymatic ligation with n different molecular barcodes to the plurality of cell-free nucleic acid molecules, wherein n is at least 2 and no more than 10,000*z, wherein z is a mean of an expected number of duplicate molecules in the plurality of cell-free nucleic acid cell-free nucleic acid molecules having identical start and stop positions.

6 . The method of claim 5 , wherein the enzymatic ligation is performed using blunt-end ligation or sticky-end ligation.

7 . The method of claim 5 , wherein n different molecular barcodes is no more than 1,000*z.

8 . The method of claim 5 , wherein n different molecular barcodes is no more than 100*z.

9 . The method of claim 1 , comprising enriching the amplified progeny polynucleotides prior to the sequencing in (c) for regions of interest.

10 . The method of claim 9 , wherein the enriching comprises selectively enriching for regions of interest that comprise genomic sequences of genes, oncogenes, tumor suppressor genes, promoters, or regulatory sequence elements.

11 . The method of claim 1 , further comprising mapping the set of sequencing reads to a reference sequence to generate mapped sequencing reads.

12 . The method of claim 5 , further comprising, mapping the set of sequencing reads to a reference sequence to generate mapped sequencing reads and prior to (d), grouping a plurality of mapped sequencing reads into families using sequence information from the molecular barcodes, a start base position of a given mapped sequence read from among the plurality of mapped sequence reads at which the given sequence read is determined to start mapping to the reference sequence and a stop base position of the given mapped sequence read at which the given sequence read is determined to stop mapping to the reference sequence.

13 . The method of claim 12 , wherein the collapsing in (d) comprises generating a consensus sequence from each family from among one or more of the families.

14 . The method of claim 1 , wherein the subject has cancer.

15 . The method of claim 1 , further comprising detecting cancer in the subject based on the one or more methylation profiles determined in (d).

16 . The method of claim 13 , further comprising identifying one or more consensus sequences comprising a somatic genetic variant as compared to the reference sequence.

17 . The method of claim 16 , wherein the somatic genetic variant comprises a single nucleotide variant (SNV), an insertion or deletion (indel), a copy number variation (CNV), or a gene fusion.

18 . The method of claim 17 , further comprising detecting cancer in the subject when the presence of the one or more somatic genetic variants is detected.

19 . The method of claim 11 , wherein the grouping sequencing reads comprises grouping based on sequence information at the beginning (start) and end (stop) regions of the sequence read and the length of the sequence read.

20 . The method of claim 1 , wherein (d) further comprises determining a quantitative measure of polymorphic forms from among a plurality of consensus sequences.

21 . The method of claim 13 , wherein (d) further comprises determining a quantitative measure of polymorphic forms from among a plurality of consensus sequences.

Assignments (1)

Continuity (22)

References Cited (400)