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Microbial Genomics logo Microbial Genomics

Volume 11, Issue 9

AmpliconTyper – a tool for analysing ONT multiplex PCR data from environmental and other complex samples Open Access

Abstract

Amplicon sequencing is a popular method for understanding the diversity of bacterial communities in samples containing multiple organisms as exemplified by 16S rRNA sequencing. Another application of amplicon sequencing includes multiplexing both primer sets and samples, allowing sequencing of multiple targets in multiple samples in the same sequencing run. Multiple tools exist to process the amplicon sequencing data produced via the short-read Illumina platform, but there are fewer options for long-read Oxford Nanopore Technologies (ONT) sequencing, or for processing data from environmental surveillance or other sources with many different organisms. We have developed AmpliconTyper (v0.1.28, DOI: 10.5281/zenodo.15045111) for analysing multiplex amplicon sequencing data from environmental (e.g. wastewater) or similarly complex samples, generated using ONT devices. The tool uses machine learning to classify sequencing reads into target and non-target organisms with very high specificity and sensitivity. The user can train models using public and/or user-generated data, which can subsequently be applied to analyse new data. The tool can also generate amplicon consensus sequences, as well as identify SNPs and report their genotype implications, such as association with lineages or antimicrobial resistance (AMR). The tool is freely available via Bioconda and GitHub (https://github.com/AntonS-bio/AmpliconTyper). AmpliconTyper allows robust identification of target organism reads in ONT-sequenced environmental samples and can identify user-specified lineage or AMR markers.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): amplicons , environmental surveillance , multiplex PCR and Oxford Nanopore Technologies
Funding
This study was supported by the:
  • Bill and Melinda Gates Foundation (Award INV047158)
    • Principal Award Recipient: NicholasGrassly
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2025-09-10
2025-12-12

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AmpliconTyper – a tool for analysing ONT multiplex PCR data from environmental and other complex samples
M Gen 11, 001421 (2025); https://doi.org/10.1099/mgen.0.001421
/content/journal/mgen/10.1099/mgen.0.001421
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