Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing
The effects of sample multiplexing on the detection sensitivity of antimicrobial resistance genes (ARGs) and pathogenic bacteria in metagenomic sequencing remain underexplored in newer sequencing technologies such as Oxford Nanopore Technologies (ONT), despite its critical importance for surveillance applications. Here, we evaluate how different multiplexing levels (four and eight samples per flowcell) on two ONT platforms, GridION and PromethION P2 Solo, influence the detection of ARGs, bacterial taxa and pathogens. While overall resistome and bacterial community profiles remained comparable across multiplexing levels, ARG detection was more comprehensive in the four-plex setting, however only with low-abundance genes. Similarly, pathogen detection was more sensitive in the four-plex, identifying a broader range of pathogens compared to the eight-plex, yet remains with low abundant taxa. However, triplicate sequencing revealed that these differences were primarily due to sequencing variability rather than multiplexing itself, as similar inconsistencies were observed across replicates. Given that eight-plex sequencing is more cost-effective while still capturing the overall resistome and bacterial community composition, it may be the preferred option for general surveillance. Conversely, lower multiplexing levels may be advantageous for applications requiring enhanced sensitivity, such as detailed pathogen surveillance. These findings highlight the trade-off between multiplexing efficiency, sequencing depth, and cost in metagenomic studies.