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 surveillanc...
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Frontiers Media S.A.
2025-06-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1597804/full |
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| author | Mirena Ivanova Frank M. Aarestrup Saria Otani |
| author_facet | Mirena Ivanova Frank M. Aarestrup Saria Otani |
| author_sort | Mirena Ivanova |
| collection | DOAJ |
| description | 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 cost-effective 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. |
| format | Article |
| id | doaj-art-c8b9b5c3d4fa40d1bfc4ee438a5d7c1c |
| institution | DOAJ |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-c8b9b5c3d4fa40d1bfc4ee438a5d7c1c2025-08-20T03:22:04ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-06-011610.3389/fmicb.2025.15978041597804Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencingMirena IvanovaFrank M. AarestrupSaria OtaniThe 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 cost-effective 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.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1597804/fullmicrobiomelong read sequencingAMRbacteriamultiplexing |
| spellingShingle | Mirena Ivanova Frank M. Aarestrup Saria Otani Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing Frontiers in Microbiology microbiome long read sequencing AMR bacteria multiplexing |
| title | Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing |
| title_full | Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing |
| title_fullStr | Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing |
| title_full_unstemmed | Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing |
| title_short | Impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long-read sequencing |
| title_sort | impact of sample multiplexing on detection of bacteria and antimicrobial resistance genes in pig microbiomes using long read sequencing |
| topic | microbiome long read sequencing AMR bacteria multiplexing |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1597804/full |
| work_keys_str_mv | AT mirenaivanova impactofsamplemultiplexingondetectionofbacteriaandantimicrobialresistancegenesinpigmicrobiomesusinglongreadsequencing AT frankmaarestrup impactofsamplemultiplexingondetectionofbacteriaandantimicrobialresistancegenesinpigmicrobiomesusinglongreadsequencing AT sariaotani impactofsamplemultiplexingondetectionofbacteriaandantimicrobialresistancegenesinpigmicrobiomesusinglongreadsequencing |