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Fast and efficient method for parallel construction of targeted exome and methylome single-stranded DNA sequencing

Eunhye Kim¹, Sinae An^1,2, Heerak Ahn²

¹Department of Pharmacy, School of Pharmacy and Institute of New Drug Development, Jeonbuk National University, Jeonju, 54907, Republic of Korea.

Scientific Reports

|February 28, 2025

Summary

We developed an efficient workflow for parallel genomic and epigenomic sequencing library construction from limited, damaged DNA. This method accurately identifies mutations and methylation in pediatric brain cancer samples, even from aged formalin-fixed tissues.

Keywords:

Exome Formalin-fixed paraffin-embedded (FFPE)Methylome Pediatric brain cancer Single-stranded DNA library

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Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Published on: May 2, 2025

Area of Science:

Genomics
Epigenomics
Cancer Research

Background:

DNA damage from formalin fixation and oxidation complicates genomic and epigenomic analysis.
Small DNA input amounts limit sequencing library construction efficiency.
Pediatric brain cancers often involve DNA damage, requiring robust analytical methods.

Purpose of the Study:

To establish an efficient workflow for parallel targeted exome and methylome sequencing library construction.
To assess the protocol's performance on damaged DNA from formalin-fixed paraffin-embedded (FFPE) pediatric brain cancer samples.
To evaluate the accuracy and uniformity of sequencing data obtained from low-input, damaged DNA.

Main Methods:

Development of a single-stranded DNA library construction protocol.
Parallel targeted exome and methylome sequencing.
Application to nine pediatric brain cancer FFPE DNA samples.
Bioinformatics filtering of artifactual mutation calls.

Main Results:

Superior exome coverage uniformity compared to previous methods.
Reproducibility of 89.4-97.0% of somatic single nucleotide variant (SNV) calls from FFPE samples with 26.7-50ng DNA input.
Obtained methylation calls for 78-92% of target CpGs with 100ng FFPE DNA, comparable to fresh frozen samples.
Recovered 39-76% of filtered SNVs from methylome data.

Conclusions:

A simple, efficient protocol for parallel exome and methylome library construction was established.
The protocol is effective for analyzing damaged DNA from FFPE tissues, even with prolonged storage.
This method enables robust genomic and epigenomic profiling of challenging clinical samples.