Methods and systems for high-depth sequencing of methylated nucleic acid
Inventors
Ariazi, Eric • Weinberg, David • Hogan, Greg • St. John, John • Pearson, Michael
Assignees
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Publication Number
US-12503728-B2
Publication Date
2025-12-23
Expiration Date
Abstract
Methods and systems provided herein address current limitations of bisulfite-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.
Core Innovation
The invention describes targeted, high-depth methylation sequencing that uses conversion-tolerant nucleic acid adapters and enzymatic cytosine conversion, followed by probe-based enrichment and sequencing. The approach is directed to methylation profiling at a pre-identified panel of CpG, CHG, or CHH loci, with sequence comparison used to determine a methylation profile or targeted methylation pattern. The partial content also describes UMI-based duplex analysis for error correction and assessment of hemi-methylation.
The supplied partial content does not include the patent’s background section and therefore does not explicitly state the problem being solved. It does state that the approach aims to preserve cfDNA fragment endpoints and length and to improve coverage and sequencing-library yield relative to bisulfite-based workflows; these are described as intended benefits, not as a separately quoted background problem.
Claims Coverage
The two independent claims cover two inventive features: methylation sequencing to determine a methylation profile, and determining a targeted methylation pattern.
Methylation sequencing and profiling
Claim 1 covers methylation sequencing of a biological-sample nucleic acid using a conversion-tolerant adapter and enzymatic cytosine conversion, with probe enrichment for a pre-identified panel of CpG, CHG, or CHH loci. The enriched sequences are determined at greater than 100× depth and compared with a reference sequence to determine a methylation profile.
Determining a targeted methylation pattern
Claim 14 covers determining a targeted methylation pattern of a biological-sample nucleic acid using a conversion-tolerant adapter and enzymatic cytosine conversion, with probe enrichment for a pre-identified panel of CpG, CHG, or CHH loci. The enriched sequences are determined at greater than 100× depth and compared with a reference sequence to determine the targeted methylation pattern.
The independent claims cover related targeted methylation sequencing approaches: one determines a methylation profile, and the other determines a targeted methylation pattern.
Stated Advantages
The methods aim to preserve cfDNA fragment endpoints and length.
The methods aim to improve coverage and sequencing-library yield relative to bisulfite-based workflows.
UMI-based duplex analysis is described for error correction and assessment of hemi-methylation.
Documented Applications
Methylation profiling of nucleic acids at a pre-identified panel of CpG, CHG, or CHH loci.
Cancer classification using methylation profiling.
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