US-20260125748-A1 - METHYLATION SEQUENCING THROUGH METHYLATION PRESERVING AMPLIFICATION WITH ERROR CORRECTION

US20260125748A1US 20260125748 A1US20260125748 A1US 20260125748A1US-20260125748-A1

Abstract

Provided herein is a DNA analysis method comprising methylation-specific amplification with error correction. Provided herein are also methods for determining the likelihood that a subject has a disease or condition, such as cancer.

Inventors

Andrew Kennedy

Assignees

GUARDANT HEALTH, INC.

Dates

Publication Date: 20260507
Application Date: 20250619

Claims (20)

1 . (canceled)
2 . A method of analyzing DNA, the method comprising: (a) performing a methylation-preserving amplification of the DNA, wherein the DNA comprises barcodes; (b) subjecting the DNA to a procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity; (c) enriching for one or more sets of epigenetic target regions of DNA from the DNA, thereby providing enriched DNA, wherein the enriching comprises contacting the DNA with target-specific probes specific for the one or more sets of epigenetic target regions; and (d) sequencing the DNA and determining an epigenetic consensus sequence of the DNA associated with at least a portion of the barcodes.
3 . A method of analyzing DNA, the method comprising: (a) performing a methylation-preserving amplification of the DNA, wherein the DNA comprises barcodes; (b) enriching for one or more sets of epigenetic target regions of DNA from the DNA, thereby providing enriched DNA, wherein the enriching comprises contacting the DNA with target-specific probes specific for the one or more sets of epigenetic target regions; (c) sequencing the enriched DNA in a modification-sensitive manner and determining an epigenetic consensus sequence of the enriched DNA associated with at least a portion of the barcodes.
4 . A method of analyzing DNA, the method comprising: (a) performing a linear, methylation-preserving amplification of the DNA; and (b) sequencing the DNA in a modification-sensitive manner and determining an epigenetic consensus sequence of the DNA.
5 . A method of analyzing DNA, the method comprising: (a) performing a linear, methylation-preserving amplification of the DNA; (b) subjecting the DNA to a procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity; and (c) sequencing the DNA and determining an epigenetic consensus sequence of the DNA.
6 . A method of analyzing DNA, the method comprising: (a) performing a linear, methylation-preserving amplification of the DNA; (b) enriching for one or more sets of epigenetic target regions of DNA from the DNA, thereby providing enriched DNA; (c) sequencing the enriched DNA in a modification-sensitive manner and determining an epigenetic consensus sequence of the enriched DNA.
7 . The method of claim 3 , wherein the sequencing in a modification-sensitive manner comprises subjecting the DNA to a procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity.
8 . The method of claim 2 , wherein (a) the DNA comprises barcodes, (b) the method comprises ligating adapters comprising barcodes to the DNA prior to the sequencing; (c) the method comprises ligating adapters comprising barcodes to the DNA prior to amplifying the DNA; and/or (d) the method comprises ligating adapters comprising barcodes to the DNA prior to performing the methylation-preserving amplification of the DNA.
9 .- 10 . (canceled)
12 . A method of analyzing DNA, the method comprising: (a) performing a methylation-preserving amplification of the DNA, wherein the DNA comprises inserts and adapters comprising barcodes, at least one of the adapters further comprises a restriction enzyme cleavage site between a barcode and a portion of the adapter, and the barcode is located between the insert and the restriction enzyme cleavage site, thereby providing amplified DNA; (b) contacting the amplified DNA with a restriction enzyme that recognizes and cleaves the DNA at the restriction enzyme cleavage site in the adapter; (c) before or after step (b), subjecting the amplified DNA to a procedure that affects a first nucleobase of the amplified DNA differently from a second nucleobase of the amplified DNA, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity; (d) after steps (b) and (c), ligating supplemental adapters to the amplified DNA, optionally wherein the supplemental adapters do not comprise barcodes; (e) after step (d), performing a uracil- and/or dihydrouracil-tolerant amplification of the DNA; (f) after step (e), enriching for one or more sets of epigenetic target regions of DNA from the amplified DNA, thereby providing enriched DNA; (g) optionally further amplifying the enriched DNA; and (h) sequencing the enriched DNA and determining an epigenetic consensus sequence of the DNA associated with at least a portion of the barcodes.
13 . The method of claim 12 , wherein the uracil- and/or dihydrouracil-tolerant amplification of the DNA comprises PCR using a uracil- and/or dihydrouracil-tolerant DNA polymerase, and/or wherein the optional step of amplifying the enriched DNA (step (g)) is performed.
14 . (canceled)
15 . The method of claim 13 , wherein the step of amplifying the enriched DNA further comprises differentially tagging the enriched DNA, optionally, wherein differentially tagging the enriched DNA comprises attaching one or more sample indices to the DNA.
16 . (canceled)
17 . The method of claim 2 , wherein the barcodes do not comprise cytosines in a non-CpG context.
18 . The method of claim 2 , wherein the DNA comprises inserts and adapters comprising barcodes, at least one of the adapters further comprises a restriction enzyme cleavage site between a barcode and a portion of the adapter, and the barcode is located between the insert and the restriction enzyme cleavage site.
19 . The method of claim 18 , further comprising contacting the amplified DNA with a restriction enzyme that recognizes and cleaves the DNA at the restriction enzyme cleavage site in the adapter.
20 . The method of claim 19 , further comprising ligating supplemental adapters to the amplified DNA (a) after the step of contacting the amplified DNA with a restriction enzyme that recognizes and cleaves the DNA at the restriction enzyme cleavage site in the adapter, and (b) prior to or after a step of subjecting the amplified DNA to a procedure that affects a first nucleobase of the amplified DNA differently from a second nucleobase of the amplified DNA, wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity; optionally wherein the supplemental adapters do not comprise barcodes.
21 . The method of claim 20 , further comprising performing a uracil- and/or dihydrouracil-tolerant amplification of the DNA, optionally wherein the uracil- and/or dihydrouracil-tolerant amplification of the DNA comprises PCR using a uracil- and/or dihydrouracil-tolerant DNA polymerase.
22 .- 23 . (canceled)

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a Continuation of International Patent Application No. PCT/US2023/085248, filed Dec. 20, 2023, which claims the benefit of priority of U.S. Provisional Patent Application No. 63/476,914, filed Dec. 22, 2022, which is incorporated by reference herein for all purposes. FIELD OF THE INVENTION The present disclosure provides compositions and methods related to analyzing DNA, such as cell-free DNA. In some embodiments, the DNA is from a subject having or suspected of having cancer and/or the DNA includes DNA from cancer cells. In some embodiments, the DNA is amplified using a methylation-preserving amplification, prior to sequencing. In some embodiments, the methylation-preserving amplification comprises a DNA methyltransferase, such as DNA methyltransferase 1 (DNMT1). INTRODUCTION AND SUMMARY Cancer is responsible for millions of deaths per year worldwide. Early detection of cancer may result in improved outcomes because early-stage cancer tends to be more susceptible to treatment. Improperly controlled cell growth is a hallmark of cancer that generally results from an accumulation of genetic and epigenetic changes, such as copy number variations (CNVs), single nucleotide variations (SNVs), gene fusions, insertions and/or deletions (indels), epigenetic variations including modification of cytosine (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, and other more oxidized forms) and association of DNA with chromatin proteins and transcription factors. Thus, cancer can be indicated by non-sequence modifications, such as methylation. Examples of methylation changes in cancer include local gains of DNA methylation in the CpG islands at the TSS of genes involved in normal growth control, DNA repair, cell cycle regulation, and/or cell differentiation. Hypermethylation can be associated with an aberrant loss of transcriptional capacity of involved genes and occurs at least as frequently as point mutations and deletions as a cause of altered gene expression. Furthermore, without wishing to be bound by any particular theory, cells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject. The DNA from such cells may differ epigenetically from shed DNA in a healthy subject. As such, the distribution of epigenetically modified (e.g., methylated) DNA in certain DNA samples, such as cell-free DNA (cfDNA), may change upon carcinogenesis. Thus, sufficiently sensitive epigenetic (e.g., DNA methylation) profiling can be used to detect aberrant methylation in DNA of a sample. Biopsies represent a traditional approach for detecting or diagnosing cancer in which cells or tissue are extracted from a possible site of cancer and analyzed for relevant phenotypic and/or genotypic features. Biopsies have the drawback of being invasive. Detection of cancer based on analysis of body fluids (“liquid biopsies”), such as blood, is an intriguing alternative based on the observation that DNA from cancer cells is released into body fluids. A liquid biopsy is noninvasive (sometimes requiring only a blood draw). However, it has been challenging to develop accurate and sensitive methods for analyzing liquid biopsy material that provides detailed information regarding nucleobase modifications given the low concentration and heterogeneity of cell-free DNA. The contribution of DNA from cells in or around a cancer or neoplasm to a sample may be relatively small relative to the contribution from other cells, and the DNA contributed from other cells may be uninformative as to cancer status. Isolating and processing the fractions of cell-free DNA useful for further analysis in liquid biopsy procedures is an important part of these methods. Further, current methods of cancer diagnostic assays of cell-free nucleic acids (e.g., cell-free DNA or cell-free RNA) may focus on the detection of tumor-related somatic variants, including single nucleotide variants (SNVs), copy number variations (CNVs), fusions, and indels (i.e., insertions or deletions), which are all mainstream targets for liquid biopsy. There is growing evidence that non-sequence modifications like methylation status and fragmentomic signal in cell-free DNA can provide information on the source of cell-free DNA and disease level. In addition, different types of modifications such as 5-methylation and 5-hydroxymethylation can have different implications as to the presence or absence of disease. Detailed knowledge of the non-sequence modifications of the cell-free DNA (e.g., when combined with somatic mutation calling) can improve assessments of tumor status. Accordingly, there is a continued need for improved methods and compositions for analyzing DNA, including cell-free DNA, e.g., in liquid biopsies. The present disclosure aims to meet the need for improved analysis of DNA, such as cell-free DNA and/or provide other benefits. In some embodiments, the present disclosure provides sin