Methods and systems for high-depth sequencing of methylated nucleic acid
AssigneeFreenome Holdings, Inc.
InventorsEric ARIAZI, David WEINBERG, Greg HOGAN, John ST. JOHN, Michael PEARSON
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Abstract
Methods and systems provided herein address current limitations of bisulfate-based methylation sequencing by improving the quality and accuracy of nucleic acid methylation sequencing and uses thereof for detection of disease. Methods that include minimally-destructive conversion methods for methylation sequencing as well as specialized UMI adapters provide for improved quality of sequencing libraries and sequencing information. Greater accuracy and more complete methylation-state information permits higher quality feature generation for use in machine learning models and classifier generation.
Claims21
A method of methylation sequencing a nucleic acid molecule of a biological sample, comprising:
- a) ligating a nucleic acid adapter comprising a unique molecular identifier to the nucleic acid molecule, wherein the nucleic acid molecule comprises unconverted nucleic acids;
- b) converting unmethylated cytosines to uracils in the nucleic acid molecule using a minimally-destructive conversion method, thereby generating converted nucleic acids;
- c) amplifying the converted nucleic acids at least in part by polymerase chain reaction, 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 or CH loci to enrich for sequences corresponding to the pre-identified panel of CpG or CH loci, thereby generating enriched converted nucleic acids;
- e) determining 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 or CH loci to determine a methylation profile of the nucleic acid molecule of the biological sample.
A method for determining a targeted methylation pattern of a nucleic acid molecule of a biological sample, comprising:
- a) ligating a nucleic acid adapter comprising a unique molecular identifier to the nucleic acid molecule, wherein the nucleic acid molecule comprises unconverted nucleic acids;
- b) enzymatically converting unmethylated cytosines to uracils in the nucleic acid molecule, thereby generating converted nucleic acids;
- c) amplifying the converted nucleic acids at least in part by polymerase chain reaction, 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 or CH loci to enrich for sequences corresponding to the pre-identified panel of CpG or CH loci, thereby generating enriched converted nucleic acids;
- e) determining 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 or CH loci to determine the targeted methylation pattern of the nucleic acid molecule of the biological sample.
-43. (canceled)
Dependencies are inferred from the claim text.