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ORIGINAL RESEARCH article

Front. Microbiol.

Sec. Infectious Agents and Disease

Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1597804

This article is part of the Research TopicInfectious disease control in the microbial functional genomics eraView all 9 articles

Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing

Provisionally accepted
  • Technical University of Denmark, Kongens Lyngby, Denmark

The final, formatted version of the article will be published soon.

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, 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 with low-abundance genes. Similarly, pathogen detection was more sensitive in the four-plex, identifying a broader range of low abundant bacterial taxa compared to the eight-plex. However, triplicate sequencing of the same microbiomes 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 costeffective while still capturing the overall resistome and bacterial community composition, it may be the preferred option for general surveillance. Lower multiplexing levels may be advantageous for applications requiring enhanced sensitivity, such as detailed pathogen research. These findings highlight the trade-off between multiplexing efficiency, sequencing depth, and cost in metagenomic studies.

Keywords: microbiome, Long read sequencing, AMR, Bacteria, multiplexing

Received: 21 Mar 2025; Accepted: 28 May 2025.

Copyright: © 2025 Ivanova, Aarestrup and Otani. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Saria Otani, Technical University of Denmark, Kongens Lyngby, Denmark

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