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

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.