Optimized bacterial community characterization through full-length 16S rRNA gene sequencing utilizing MinION nanopore technology.

Bertolo, Alessandro; Valido, Ezra; Stoyanov, Jivko (2024). Optimized bacterial community characterization through full-length 16S rRNA gene sequencing utilizing MinION nanopore technology. BMC microbiology, 24(1), p. 58. BioMed Central 10.1186/s12866-024-03208-5

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Accurate identification of bacterial communities is crucial for research applications, diagnostics, and clinical interventions. Although 16S ribosomal RNA (rRNA) gene sequencing is a widely employed technique for bacterial taxonomic classification, it often results in misclassified or unclassified bacterial taxa. This study sought to refine the full-length 16S rRNA gene sequencing protocol using the MinION sequencer, focusing on the V1-V9 regions. Our methodological enquiry examined several factors, including the number of PCR amplification cycles, choice of primers and Taq polymerase, and specific sequence databases and workflows employed. We used a microbial standard comprising eight bacterial strains (five gram-positive and three gram-negative) in known proportions as a validation control.


Based on the MinION protocol, we employed the microbial standard as the DNA template for the 16S rRNA gene amplicon sequencing procedure. Our analysis showed that an elevated number of PCR amplification cycles introduced PCR bias, and the selection of Taq polymerase and primer sets significantly affected the subsequent analysis. Bacterial identification at genus level demonstrated Pearson correlation coefficients ranging from 0.73 to 0.79 when assessed using BugSeq, Kraken-Silva and EPI2ME-16S workflows. Notably, the EPI2ME-16S workflow exhibited the highest Pearson correlation with the microbial standard, minimised misclassification, and increased alignment accuracy. At the species taxonomic level, the BugSeq workflow was superior, with a Pearson correlation coefficient of 0.92.


These findings emphasise the importance of careful selection of PCR settings and a well-structured analytical framework for 16S rRNA full-length gene sequencing. The results showed a robust correlation between the predicted and observed bacterial abundances at both the genus and species taxonomic levels, making these findings applicable across diverse research contexts and with clinical utility for reliable pathogen identification.

Item Type:

Journal Article (Original Article)


04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Social and Preventive Medicine (ISPM)
04 Faculty of Medicine > Department of Orthopaedic, Plastic and Hand Surgery (DOPH) > Clinic of Orthopaedic Surgery

UniBE Contributor:

Bertolo, Alessandro, Stoyanov, Jivko


600 Technology > 610 Medicine & health
300 Social sciences, sociology & anthropology > 360 Social problems & social services




BioMed Central


[223] Swiss Paraplegic Foundation = Schweizer Paraplegiker-Stiftung ; [256] Swiss Paraplegic Research = Schweizer Paraplegiker-Forschung




Pubmed Import

Date Deposited:

19 Feb 2024 10:41

Last Modified:

27 Feb 2024 14:43

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PubMed ID:


Uncontrolled Keywords:

16S rRNA gene-based sequencing Bacterial DNA Nanopore sequencing V1–V9 region





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