Direct methylation single-base resolution sequencing of enriched unmethylated dna
AssigneeGuardant Health, Inc.
InventorsAndrew KENNEDY, James GHADIALI
Life of this patent
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Abstract
Described herein are methods and composition related to detection of unmethylated DNA regions with high sensitivity and low costs compared to current methods. This approach involves tagging CpG in unmethylated DNA with reagents that provides both handle for immobilization/enrichments, and blocking of CpG deamination by deaminases (e.g. APOBEC3A). By leveraging methods involving methyltransferase-based transfer of unnatural substrates to unmodified cytosines (in CpGs only), one can adapt such steps into various workflow sequences involving hyper-and hypo-methylated DNA with flexible deployment of tagging steps as suited for the analyte of interest.
Claims34
A method for detecting the methylation profile of nucleic acids in a sample, wherein the method comprises:
- partitioning the nucleic acids based on the presence or absence of 5-hydroxymethylcytosine (5hmC) nucleic acid bases in the nucleic acids;
- subjecting the nucleic acids to a conversion procedure that selectively converts the base pairing specificity of 5-methylcytosines (5mC) or unmethylated cytosines (C) in the nucleic acids;
- amplifying the nucleic acids which to generate amplification products;
- sequencing the amplification products to obtain sequencing data;
- and
- analysing the sequencing data to determine whether the cytosine nucleic acid bases of the nucleic acids in the sample are 5hmC, 5mC or C.
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Dependencies are inferred from the claim text.