Genome-wide methylation detection and episignature analysis using PacBio long-read sequencing

Area of Science:

• Genomics
• Epigenetics
• Molecular Biology

Background:

• Accurate detection of 5-methylcytosine (5mC) patterns is crucial for diagnosing genetic conditions.
• Genome-wide methylation episignatures offer a novel approach for resolving variants of uncertain significance.
• Traditional methods like short-read bisulfite sequencing and methylation arrays have limitations in comprehensive DNA methylation detection.

Purpose of the Study:

• To assess the capability of Pacific Biosciences (PacBio) long-read sequencing (LRS) for robust, genome-wide methylation detection for medical applications.
• To evaluate PacBio LRS in identifying differentially methylated imprinted regions and genome-wide epigenetic signatures relevant to genetic disorders.

Main Methods:

• PacBio LRS methylation detection was benchmarked against two conventional genome-wide DNA methylation sequencing techniques.
• Analysis involved 30 PacBio LRS samples (10 trios at ~30× coverage) to assess differential methylation in 25 known parentally imprinted regions.
• Two published epigenetic signatures for KMT2A gene defects were evaluated using PacBio LRS data from KMT2A patients, VUS samples, and controls.

Main Results:

• PacBio methylation calls demonstrated high concordance with short-read protocols, yielding 5% more calls, particularly in challenging regions.
• Haplotype-resolved methylation patterns were accurately identified in 96% of imprinted regions, with correct parental origin confirmation.
• Downsampling indicated robust detection of imprinted regions at a minimum of 15× genome-wide coverage.
• Both evaluated KMT2A episignatures successfully distinguished KMT2A patients from controls and classified VUS samples as controls.

Conclusions:

• PacBio LRS reliably detects specific, medically relevant methylation changes.
• The technology effectively identifies genome-wide episignatures in rare disease patients.
• PacBio LRS represents a promising tool for advancing the diagnosis and understanding of genetic conditions through methylation analysis.