Full-length 16S rRNA gene nanopore sequencing for bacterial identification in routine diagnostics

Introduction: Culture-negative bacterial infections can result from various factors, including prior antibiotic therapy, slow-growing bacteria, or extended transport times. In such cases, 16S rRNA gene Sanger sequencing can be employed to identify uncultured bacteria. However, Sanger sequencing is limited to monobacterial infections and may require significant time for results. Advances in sequencing technologies, such as nanopore sequencing, offer potential benefits for species identification in polybacterial infections, expanded species identification, and in-house sequencing capabilities.

Goals: The primary objectives of this study were to assess the accuracy and efficacy of full-length 16S rRNA gene nanopore sequencing in bacterial species identification, comparing it to the established Sanger sequencing method. Additionally, we aimed to evaluate whether nanopore sequencing could provide enhanced resolution for specific microbial species.

Materials & Methods: Forty samples, cultured and sequenced with Sanger in routine diagnostics, were selected for this comparative study. Subsequently, these samples underwent nanopore sequencing, and the results were compared among the three methods.

Results: Our findings demonstrated a high level of concordance between Sanger and nanopore sequencing for species identification, including the resolution of mixed sequences in polybacterial infections. Nanopore sequencing successfully identified certain species that would have remained undetected using Sanger sequencing.

Summary: In conclusion, this study highlights nanopore sequencing as a potentially valuable tool, especially in cases of culture-negative infections and critically ill patients where urgent bacteriological diagnosis is crucial. Further exploration and validation of nanopore sequencing in larger cohorts should be pursued to comprehensively assess its utility and integration into routine diagnostic workflows.