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

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.