Different DNA input amounts and library preparation kits yield comparable resistome characterization using target-enriched metagenomic sequencing.

Introduction: Target-enriched metagenomic sequencing improves detection of antimicrobial resistance genes (ARGs) relative to standard shotgun metagenomics, but whether DNA input amount influences resistome characterization has not been systematically evaluated. Methods: We evaluated target-enriched sequencing using fecal microbial communities from multiple host species and compared results across library preparation kits, DNA input amounts, and sequencing approaches. Composite fecal samples from livestock and humans and individual canine samples were processed using three library preparation kits and DNA input amounts ranging from 50 to 800 ng. Results: Target-enriched libraries from composite samples were compared with shotgun metagenomic sequences generated from the same samples. Target-enriched sequencing achieved a 7.9-fold increase in on-target read proportions and detected 12-fold more unique antimicrobial resistance gene groups than shotgun metagenomics. Resistome composition was driven primarily by sample source, which explained 66%-79% of within-species variance, while library preparation kit accounted for 13%-26% and DNA input amount had no consistent effect. Kit-associated differences were systematic and affected primarily low-abundance resistance classes but were small relative to biological variation among samples. Discussion: These findings demonstrate that target-enriched metagenomic sequencing substantially improves resistome detection and provides comparable resistome characterization DNA input amounts ranging from 50 to 800 ng. Differences associated with library preparation were small relative to biological variation, supporting the flexibility of using differing DNA inputs for resistome characterization.