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<p>The detection and identification of bacterial species in clinical samples are crucial for patient management and antibiotic treatment. When culture-based identification methods fail, 16S rRNA gene next-generation sequencing (NGS) serves as a valuable alternative. However, its clinical utility is often limited by prolonged time to results (TtR) and limited species-level resolution. This study aimed to develop and validate a faster, more discriminative 16S rRNA gene NGS workflow. Our current 16S rRNA gene NGS protocol uses micelle-based PCR (micPCR) targeting the V4 region, followed by Illumina sequencing. This method ensures accurate quantification of 16S rRNA gene copies in (low biomass) clinical samples by reducing PCR artefacts and correcting for background DNA contamination. To shorten the TtR and improve species-level determination, the micPCR protocol was adapted to amplify full-length 16s rRNA genes, followed by nanopore sequencing using the Flongle Flow Cell with automated data analysis using the Genome Detective platform. Testing with a synthetic microbial community and six clinical samples showed that the 16S rRNA gene micPCR/nanopore sequencing protocol maintains good accuracy and sensitivity, reducing TtR to 24&#xa0;h and enhancing species-level resolution. This optimized workflow improves clinical diagnostics, making it a valuable tool for guiding patient treatment decisions.</p>

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Rapid and reliable species-level identification from clinical samples using 16S rRNA gene nanopore sequencing analysis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月5日

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  • Marco J. R. Ivens,
  • Sergio Kamminga,
  • Kawtar Benchamach,
  • Chaimae Akile,
  • Els Wessels,
  • Eric C. J. Claas,
  • Stefan A. Boers

摘要

The detection and identification of bacterial species in clinical samples are crucial for patient management and antibiotic treatment. When culture-based identification methods fail, 16S rRNA gene next-generation sequencing (NGS) serves as a valuable alternative. However, its clinical utility is often limited by prolonged time to results (TtR) and limited species-level resolution. This study aimed to develop and validate a faster, more discriminative 16S rRNA gene NGS workflow. Our current 16S rRNA gene NGS protocol uses micelle-based PCR (micPCR) targeting the V4 region, followed by Illumina sequencing. This method ensures accurate quantification of 16S rRNA gene copies in (low biomass) clinical samples by reducing PCR artefacts and correcting for background DNA contamination. To shorten the TtR and improve species-level determination, the micPCR protocol was adapted to amplify full-length 16s rRNA genes, followed by nanopore sequencing using the Flongle Flow Cell with automated data analysis using the Genome Detective platform. Testing with a synthetic microbial community and six clinical samples showed that the 16S rRNA gene micPCR/nanopore sequencing protocol maintains good accuracy and sensitivity, reducing TtR to 24 h and enhancing species-level resolution. This optimized workflow improves clinical diagnostics, making it a valuable tool for guiding patient treatment decisions.