Optimized Bacterial Community Characterization through Full-length 16S rRNA Gene sequencing utilizing MinION Nanopore Technology

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