Enriching Microbial Cell-Free DNA in Clinical Metagenomics Using Epigenetic Filters

Area of Science:

• Genomics
• Molecular Biology
• Infectious Disease Diagnostics

Background:

• Noninvasive cell-free DNA (cfDNA) metagenomic sequencing offers hypothesis-free pathogen detection.
• Clinical sensitivity is limited by high levels of host cfDNA obscuring microbial signals.
• Differences in DNA methylation between microbial and human genomes present an opportunity for enrichment.

Purpose of the Study:

• To develop and evaluate an epigenetically guided strategy for enriching microbial cfDNA.
• To improve the signal-to-background ratio in metagenomic sequencing for pathogen detection.

Main Methods:

• Developed Epigenetically filtered Metagenomic Sequencing (EpiMeta-seq) using the methylation-sensitive enzyme HpaII.
• HpaII selectively digests unmethylated CCGG sites prevalent in microbial DNA but not human DNA.
• Enriched cfDNA fragments were sequenced and analyzed using metagenomic informatics pipelines.

Main Results:

• EpiMeta-seq achieved mean enrichment factors of 24.5-fold for fungi and 11.4-fold for bacteria in spike-in experiments.
• Clinical samples showed an average 10.0-fold increase in microbial reads per million.
• Viral DNA exhibited the highest enrichment (mean 11.5-fold), with bacterial enrichment varying by species.

Conclusions:

• EpiMeta-seq effectively enriches microbial cfDNA by exploiting host-microbe methylation differences.
• This proof-of-concept strategy enhances the microbial cfDNA signal-to-background ratio.
• EpiMeta-seq shows potential for improving pathogen detection across diverse microbial types in metagenomic sequencing.