Long-read sequencing reveals putatively mobilizable resistance genes and multi-drug resistance plasmids underestimated by short-read metagenomics.

While shotgun metagenomics is often used to profile antibiotic resistome in gut microbial communities, few studies have investigated if the choice of sequencing platform and assembly strategy affect what mobile genetic elements and antimicrobial resistance genes are recovered. In this study, we compared three platforms (Illumina, Oxford Nanopore, and PacBio HiFi) and seven assembly strategies on gut metagenomes from cattle, pig, and human as case studies. Long-read assemblies recovered 5- to 7-fold more plasmid sequence than Illumina in cattle and pig (mean 17.0 Mb vs. 3.1 Mb), while Illumina performed comparably in the less diverse human gut where high per-species coverage enabled effective short-read plasmid assembly. Long reads also detected more resistance genes on plasmid contigs. Hybrid assembly results depended on the algorithm: scaffolding-based OPERA-MS preserved long-read contiguity and recovered more plasmid-borne resistance genes, while the short-read-centric metaSPAdes hybrid mode produced fragmented assemblies. After collapsing haplotype redundancy, PacBio HiFi identified 2 and 49 unique multi-drug resistance plasmid lineages in cattle and pig, respectively. On the other hand, only 2 and 4 were identified from Illumina. Long reads also placed far more ARGs in a putative mobilization context (50-73%) compared to 14-21% for short reads. Platform and assembly strategy are thus key variables in mobilome and resistome characterization and should be accounted for in antimicrobial resistance surveillance.