Journal Article

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

Bertolo, Alessandro, Stoyanov, Jivko, Valido, Ezra

2024

Overview

Background 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. Results 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. Conclusions 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: 16S rRNA gene-based sequencing / Amplification / Analysis / Bacteria / Bacteria - genetics / Bacterial DNA / Bar codes / Bioinformatics / Biological Microscopy / Biomedical and Life Sciences / Correlation coefficient / Correlation coefficients / DNA / DNA polymerase / DNA sequencing / DNA, Bacterial - genetics / Gene sequencing / Genes / Genes, rRNA / Genetic testing / Health aspects / High-Throughput Nucleotide Sequencing - methods / Life Sciences / Mathematical analysis / Microbiology / Microbiota / Microbiota (Symbiotic organisms) / Microorganisms / Mycology / Nanopore sequencing / Nanopores / Nucleotide sequencing / Parasitology / Pathogens / Polymerase chain reaction / Protocol / Ribosomal RNA / RNA, Ribosomal, 16S - genetics / rRNA 16S / Sequence Analysis, DNA - methods / Taq Polymerase - genetics / Taxonomy / Technology application / V1–V9 region / Virology / Workflow