Enriching Microbial cell-free DNA in Clinical Metagenomics using Epigenetic Filters

EpiMeta-seq uses the methylation-sensitive restriction enzyme HpaII to selectively digest unmethylated CCGG sites, which are prevalent in microbial genomes but largely methylated in human DNA. Only fragments cleaved once at unmethylated sites are incorporated into sequencing libraries, thereby enriching microbial cfDNA prior to sequencing. We assessed plasma samples from patients with microbiologically confirmed infections. Metagenomics informatics involved alignment, removal of host DNA, and taxonomic classification of sequencing reads to a curated reference database.