Analysis of methylated DNA comprising methylation-sensitive or methylation-dependent restrictions

Pending Publication Date: 2022-05-19
GUARDANT HEALTH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0003]Cancer is responsible for millions of deaths per year worldwide. Early detection of cancer ma

Problems solved by technology

Biopsies have the drawback of being invasive.
However, it has been challenging to develop accurate and sensitive methods for analyzing liquid biop

Method used

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  • Analysis of methylated DNA comprising methylation-sensitive or methylation-dependent restrictions
  • Analysis of methylated DNA comprising methylation-sensitive or methylation-dependent restrictions
  • Analysis of methylated DNA comprising methylation-sensitive or methylation-dependent restrictions

Examples

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Example

Example 1: Analysis of cfDNA to Detect the Presence / Absence of Tumor

[0494]A set of patient samples are analyzed by a blood-based NGS assay at Guardant Health (Redwood City, Calif., USA) to detect the presence / absence of cancer. cfDNA is extracted from the plasma of these patients. cfDNA of the patient samples is then combined with methyl binding domain (MBD) buffers and magnetic beads conjugated with an MBD protein and incubated overnight. Methylated cfDNA (if present, in the cfDNA sample) is bound to the MBD protein during this incubation. Non-methylated or less methylated DNA is washed away from the beads with buffers containing increasing concentrations of salt. Finally, a high salt buffer is used to wash the heavily methylated DNA away from the MBD protein. These washes result in three partitions (hypomethylated, residual methylation and hypermethylated partitions) of increasingly methylated cfDNA.

[0495]Optionally, the cfDNA molecules in the hypermethylated partition are subject...

Example

Example 2: Analysis of Methylation at Single Nucleotide Resolution in cfDNA Samples from Healthy Subjects and Subjects with Early-Stage Colorectal Cancer

[0501]Samples of cfDNA from healthy subjects and subjects with early-stage colorectal cancer were analyzed as follows. cfDNA was partitioned using MBD to provide a hypermethylated partition, an intermediate partition, and a hypomethylated partition. The partitioned DNA of each partition was ligated to adapters and subjected to an EM-seq conversion procedure whereby unmodified cytosines, but not mC and hmC, undergo deamination, although in an alternative procedure the partitioned DNA of the hypomethylated partition could be contacted with a methylation-dependent nuclease as described herein. Following such deamination, the partitions were prepared for sequencing and subjected to whole-genome sequencing. Each partition was sequenced separately, although in an alternative procedure the partitions could be differentially tagged (e.g., a...

Example

Example 3: Reduction of Technical Noise by Digestion of Nonspecifically Partitioned DNA

[0503]A pool of cfDNA from two healthy normal samples was combined, from which 18.6 ng was used as input to a MBD-partitioning assay described herein. To a subset of the samples, cfDNA from a colorectal cancer sample (CRC) with 0.5% MAF (mutant allele fraction) was added, resulting in a diluted CRC sample with 0.16% MAF. Three sets of normal samples and diluted CRC samples were used in the assay. The three sets of samples were then partitioned using MBD protein into three partitions (hyper, residual and hypo partitions). Following cleanup, the cfDNA molecules in each partition was ligated with partition-specific adapters comprising molecular barcodes. The molecular barcodes use in hyper and residual partition are selected such that they do not have MSRE recognition sites, so they are not digested in the downstream processing (irrespective of cfDNA methylation state). Post-ligation, ligation cleanu...

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Abstract

The present disclosure provides compositions and methods related to analyzing DNA, such as cell-free DNA. In some embodiments, the cell-free DNA is from a subject having or suspected of having cancer and/or the cell-free DNA includes DNA from cancer cells. In some embodiments, the DNA is partitioned into a first subsample and a second subsample, wherein the first subsample comprises DNA with a nucleotide modification (e.g., a cytosine modification) in a greater proportion than the second subsample, and the second subsample is contacted with a methylation-dependent nuclease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 086,000, filed Sep. 30, 2020, and U.S. Provisional Patent Application No. 63 / 105,183, filed Oct. 23, 2020, each of which is incorporated by reference herein in its entirety for all purposes.FIELD OF THE INVENTION[0002]The present disclosure provides compositions and methods related to analyzing DNA, such as cell-free DNA. In some embodiments, the cell-free DNA is from a subject having or suspected of having cancer and / or the cell-free DNA includes DNA from cancer cells. In some embodiments, the DNA is partitioned into a first subsample and a second subsample, wherein the first subsample comprises DNA with a nucleotide modification (e.g., a cytosine modification) in a greater proportion than the second subsample, and the second subsample is contacted with a methylation-dependent nuclease.INTRODUCTION AND SUMMARY[0003]Cancer is responsible for mill...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/10C12N9/22
CPCC12Q1/6886C12N15/1065C12N9/22C12N2800/80C12Q2600/156C12Q2600/112C12Q2600/166C12Q2600/154C12Q1/6804C12Q1/6869C12Q2535/122C12Q2537/159C12Q2563/179C12Q1/6809C12Q1/683
Inventor KENNEDY, ANDREWGREENLEAF, WILLIAM J.
Owner GUARDANT HEALTH
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