IP Library › Granted Patent US 12,565,674
Granted Patent B2
US 12,565,674 · App. 19/029,558 · Granted Mar 3, 2026

Methods and systems for analyzing nucleic acid molecules

Inventors: Andrew Kennedy (San Diego, CA); Stefanie Ann Ward Mortimer (Morgan Hill, CA); Helmy Eltoukhy (Atherton, CA); AmirAli Talasaz (Atherton, CA)
Assignee: Guardant Health, Inc.
C12Q1/6806C12N15/1065C12Q1/6874C12Q1/6886C12Q2600/154
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Quick Facts
Patent No.
US 12,565,674
App. No.
19/029,558
Granted
Mar 3, 2026
Kind
B2
Abstract

The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and/or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and/or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.

Claims (24)

1 . A method comprising:

a) providing one or more populations of cell-free DNA from a biological sample from a subject;

b) fractionating the one or more populations of cell-free DNA based on a characteristic that is present at different levels in cell-free DNA derived from cancerous compared to noncancerous cells, thereby generating subpopulations of cell-free DNA, wherein the characteristic is the methylation level of the cell-free DNA, wherein the fractionating is performed using a methyl-binding domain (MBD) protein;

c) amplifying at least one of the subpopulations of cell-free DNA;

d) sequencing at least one of the amplified subpopulations of cell-free DNA to generate sequencing data; and

e) using the sequencing data with a trained classifier to classify the characteristic as associated with a tissue in the subject, wherein the tissue is indicative of cancer and wherein the trained classifier is trained on data from subjects who have cancer, subjects who do not have cancer, and/or subjects who have different types of cancer.

2 . The method of claim 1 , wherein the trained classifier is a supervised machine learning algorithm.

3 . The method of claim 1 , wherein the biological sample is plasma.

4 . The method of claim 3 , wherein the biological sample is 0.4-40 ml of plasma.

5 . The method of claim 1 , wherein the subject has cancer or has been diagnosed as being susceptible to having cancer.

6 . The method of claim 5 , wherein the cancer is lung cancer.

7 . The method of claim 1 , wherein the MBD protein comprises MBD2.

8 . The method of claim 1 , wherein adapters are ligated to the cell-free DNA.

9 . The method of claim 8 , wherein the adapters comprise hairpin adapters.

10 . The method of claim 8 , wherein the adapters comprise molecular tags.

11 . The method of claim 8 , wherein the adapters comprise primer binding sites.

12 . The method of claim 1 , wherein the method further comprises using the trained classifier to classify the subject based on the characteristic.

13 . The method of claim 1 , wherein the trained classifier classifies the characteristic as associated with a type of cancer in the subject.

14 . The method of claim 1 , wherein the characteristic is indicative of gene expression or status of a disease.

15 . The method of claim 1 , wherein profiles of methylation level over time are used in the selection of a therapy for the subject.

16 . The method of claim 1 , wherein the method comprises monitoring residual disease or recurrence of disease.

17 . The method of claim 1 , wherein the method comprises determining the efficacy of a particular treatment option.

18 . The method of claim 1 , wherein the cell-free DNA comprises circulating tumor DNA.

19 . The method of claim 1 , wherein the methylation level is the 5′-methylcytosine level.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 23, 2026
From: MORTIMER, STEFANIE ANN WARD; TALASAZ, AMIRALI; ELTOUKHY, HELMY
To: GUARDANT HEALTH, INC.
Reel/Frame 073568/0144 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jan 24, 2025
From: KENNEDY, ANDREW
To: GUARDANT HEALTH, INC.
Reel/Frame 069999/0256 →
Continuity (9)
Continuation 18770271 · Jul 11, 2024
Continuation 18625882 · Apr 3, 2024
Continuation 18061898 · Dec 5, 2022
Continuation 16450918 · Jun 24, 2019
Continuation PCTUS2017068329 · Dec 22, 2017
Provisional Application 62550540 · Aug 25, 2017
Provisional Application 62512936 · May 31, 2017
Provisional Application 62438240 · Dec 22, 2016
Related Publication 20250171826A1 · May 29, 2025
References Cited (81)
US 6582908B2 · Fodor et al. · 2003 [cited by applicant]
US 7537898B2 · Bost et al. · 2009 [cited by applicant]
US 8476013B2 · Ehrich et al. · 2013 [cited by applicant]
US 9598731B2 · Talasaz · 2017 [cited by applicant]
US 9850523B1 · Chudova et al. · 2017 [cited by applicant]
US 10612088B2 · Shishkin et al. · 2020 [cited by applicant]
US 20010053519A1 · Fodor et al. · 2001 [cited by applicant]
US 20030152490A1 · Trulson et al. · 2003 [cited by applicant]
US 20100105049A1 · Ehrich et al. · 2010 [cited by applicant]
US 20100273165A1 · Ehrich et al. · 2010 [cited by applicant]
US 20110160078A1 · Fodor et al. · 2011 [cited by applicant]
US 20130157266A1 · Hanna et al. · 2013 [cited by applicant]
US 20140080715A1 · Lo et al. · 2014 [cited by applicant]
US 20140363815A1 · Dahl et al. · 2014 [cited by applicant]
US 20150044687A1 · Schmitt et al. · 2015 [cited by applicant]
US 20150197801A1 · Muir et al. · 2015 [cited by applicant]
US 20150299812A1 · Talasaz · 2015 [cited by applicant]
US 20160047001A1 · Larisch et al. · 2016 [cited by applicant]
US 20160201142A1 · Lo et al. · 2016 [cited by applicant]
US 20180120304A1 · Rao et al. · 2018 [cited by applicant]
US 20180251848A1 · Diehn et al. · 2018 [cited by applicant]
CN 102648292A · 2012 [cited by applicant]
EP 2805769A1 · 2014 [cited by applicant]
JP 2015521862A · 2015 [cited by applicant]
WO 2011034631A1 · 2011 [cited by applicant]
WO 2015061359A1 · 2015 [cited by applicant]
WO 2015159292A2 · 2015 [cited by applicant]
WO 2016015058A2 · 2016 [cited by applicant]
WO 2016115530A1 · 2016 [cited by applicant]
WO 2016176091A1 · 2016 [cited by applicant]
WO 2017181161A1 · 2017 [cited by applicant]
WO 2017184707A1 · 2017 [cited by applicant]
WO 2017190215A1 · 2017 [cited by applicant]
WO 2018005811A1 · 2018 [cited by applicant]
WO 2018009723A1 · 2018 [cited by applicant]
WO 2019010564A1 · 2019 [cited by applicant]
Bock, C. et al. “Quantitative comparison of genome-wide DNA methylation mapping technologies” Nature Biotech (2010) 28:1106-1114. [cited by applicant]
Burnham, P. et al. “Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma” Sci Reports (Jun. 14, 2016) 6(1), XP055472868, DOI: 10.1038/srep27859. [cited by applicant]
Chinese Office Action dated Nov. 1, 2023 for CN2017800871305. [cited by applicant]
Clark, T.A. et al. “Analytical Validation of a Hybrid Capture Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA,” J. Mol. Diagnostics (2018) 20(5):686-702. [cited by applicant]
Dey, S.S. et al. “Integrated genome and transcriptome sequencing of the same cell” Nature Biotech (Jan. 19, 2015) 33(3):285-589. [cited by applicant]
European Exam Report dated Aug. 20, 2020 for 17832453.9. [cited by applicant]
European Exam Report dated for 17832453.9, dated Aug. 5, 2022. [cited by applicant]
European Exam Report for EP Application No. 17832453.9, dated Oct. 24, 2024. [cited by applicant]
Gansauge, M-T. et al. “Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA” Nature Protocols (2013) 3:737-748. [cited by applicant]
Indian Office Action for IN Application No. 201917024437, dated Sep. 16, 2021. [cited by applicant]
International search report and written opinion dated Jul. 9, 2019 for PCT/US2017/068329. [cited by applicant]
Iurlaro, M. et al. “A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation” Genome Biology (2013) 14:R119. [cited by applicant]
Japanese office action dated Oct. 4, 2022 for JP2019-533331. [cited by applicant]
Japanese office action dated Nov. 11, 2024 for JP2023-60942. [cited by applicant]
Japanese office action dated Nov. 30, 2021 for JP2019-533331. [cited by applicant]
Jeong, H.M. et al. “Efficiency of methylated DNA immunoprecipitation bisulphite sequencing for whole-genome DNA methylation analysis” Epigenomics (2016) 8(8):1061-1077. [cited by applicant]
Jin, H. et al. “Circulating Methylated DNA as Biomarkers for Cancer Detection” Methylation: From DNA, RNA and Histones to Diseases and Treatment (2012) InTech. XP055479454, DOI:10.5772./51419. [cited by applicant]
Korean Notice of Preliminary Rejection for KR Application No. 10-2019-7020828 dated Sep. 21, 2022. [cited by applicant]
Nair, S. et al. “Enzymatic cleavage of uracil-containing single-stranded DNA linkers for the efficient release of affinity-selected circulating tumor cells” Chem Commun (2015) 51(15):3266-3269. [cited by applicant]
Newman, A. et al. “Integrated digital error suppression for improved detection of circulating tumor DNA” Nature Biotech (2016) 34(5):547-555. [cited by applicant]
Newman, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med. May 2014;20(5):548-54. doi: 10.1038/nm.3519. Epub Apr. 6, 2014. [cited by applicant]
Niazi, U. et al. “Dismiss: detection of stranded methylation in MeDIP-Seq data” BMC Bioinformatics (2016) 17:295 12 pages. [cited by applicant]
Non-final Office Action for U.S. Appl. No. 18/770,298, mailed Nov. 22, 2024. [cited by applicant]
Paweletz, C.P. et al. “Bias-corrected targeted next-generation sequencing for rapid, multiplexed detection of actionable alterations in cell-free DNA from advanced lung cancer patients” Clin Canc Res (2016) 22(4):915-92… [cited by applicant]
Phallen, J. et al. “Direct detection of early-stage cancers using circulating tumor DNA” Sci Trans Med (2017) vol. 9, Issue 403, eaan2415DOI: 10.1126/scitranslmed.aan2415. [cited by applicant]
Reuter, J.A. et al. “Simul-seq: combined DNA and RNA sequencing for whole-genome and transcriptome profiling”, Nature Methods (Oct. 10, 2016) 13(11):953-958. [cited by applicant]
Riebler, A. et al. “BayMeth: improved DNA methylation quantification for affinity capture sequencing data using a flexible Bayesian approach” Genome Biology (2014) 15:R35 19 pages. [cited by applicant]
Robinson, M.D. et al. “Evaluation of affinity-based genome-wide DNA methylation data: Effects of CpG density, amplification bias, and copy No. variation” Genome Research (2010) 20(12):1719-1729. [cited by applicant]
Rohland, N. et al. “Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture” Genome Res (2012) 22(5):939-946. [cited by applicant]
Singapore Office Action for SG application No. 11201904597S, dated Oct. 5, 2020. [cited by applicant]
Smallwood, S.A. et al. “Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity” Nature Methods (2014) 11(8):817-820. [cited by applicant]
Song, C-X. et al. “Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine” Nature Biotech (2011) 29:68-72. [cited by applicant]
Stirzaker, C. et al. “Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value” Nature Comm (2015) 6(5899) (15 pages). [cited by applicant]
Suzuki, H. et al. “Genome-wide Profiling of Chromatin Signatures Reveals Epigenetic Regulation of MicroRNA Genes in Colorectal Cancer” Cancer Research (2011) 71(17):5646-5658. [cited by applicant]
Thakur, B.K. et al. “Double-stranded DNA in exosomes: a novel biomarker in cancer detection” Cell Research—Xibao Yanjiu (Apr. 8, 2014) 24(6):766-769. [cited by applicant]
Warton, K. et al. “Methylation of cell-free circulating DNA in the diagnosis of cancer” Frontiers in Mol Biosciences (2015) 2(13), 10 pages. [cited by applicant]
Warton, K. et al. “Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma” BMC Genomics (2014) 15:476 13 pages. [cited by applicant]
Wielscher, M. et al. “Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing” BMC Clin Path (2011) 11:11 (9 pages). [cited by applicant]
Yigit, E. et al. “Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues” Appl Plant Sci (2014) 2(11):1400064. [cited by applicant]
Zhao, Y. et al. “Methylcap-Seq Reveals Novel DNA Methylation Markers for the Diagnosis and Recurrence Prediction of Bladder Cancer in a Chinese Population” PLoS One 7(4):e35175 (12 pages), (2012). [cited by applicant]
[cited by applicant]
Non-final Office Action for Canadian Patent Application No. 3046007, dated May 22, 2025. [cited by applicant]
Non-final Office Action for U.S. Appl. No. 18/770,271, mailed Nov. 18, 2024. [cited by applicant]
Non-final Office Action for U.S. Appl. No. 19/025,566, dated Mar. 12, 2025. [cited by applicant]
Defendant Tempus AI, Inc's. Opening Brief in Support of its Motion to Dismiss Patents Asserted in CA No. 25-1013 Pursuant to Federal Rule of Civil Procedure 12(b)(6) dated Dec. 3, 2025. [cited by applicant]