Methods and systems for high-depth sequencing of methylated nucleic acid

A method of methylation sequencing a nucleic acid molecule of a biological sample, comprising:

• (a) ligating a nucleic acid adapter to the nucleic acid molecule, wherein the nucleic acid molecule comprises unconverted nucleic acids, wherein the nucleic acid adapter comprises a duplex sequencing adapter;
• (b) converting unmethylated cytosines to uracils in the nucleic acid molecule using an enzymatic conversion method, thereby generating converted nucleic acids;
• (c) amplifying the converted nucleic acids, thereby generating amplified converted nucleic acids;
• (d) contacting the amplified converted nucleic acids with nucleic acid probes that are at least partially complementary to a pre-identified panel of CpG, CHG, or CHH loci to enrich for sequences corresponding to the pre-identified panel of CpG, CHG, or CHH loci, thereby generating enriched converted nucleic acids;
• (e) determining, using a duplex sequencing assay, a nucleic acid sequence of the enriched converted nucleic acids at a depth of greater than 100×;
• and
• (f) comparing the nucleic acid sequence of the enriched converted nucleic acids to a reference nucleic acid sequence of the pre-identified panel of CpG, CHG, or CHH loci to determine a methylation profile of the nucleic acid molecule of the biological sample.