Next Generation Sequencing Improves Diagnostic 16S rRNA Amplicon-Based Microbiota Analyses of Clinical Samples Compared to Sanger Sequencing

Abstract

Sequencing of the 16S ribosomal RNA (rRNA) gene is an important tool in addition to conventional methods for the identification of bacterial pathogens in human infections. In polymicrobial samples, Sanger sequencing can produce uninterpretable chromatograms. This limitation can be overcome by Next Generation Sequencing (NGS) of the 16S rRNA gene. We investigated the applicability of Oxford Nanopore Technologies (ONT) sequencing of the partial 16S rRNA gene as a diagnostic routine method for pathogen detection in clinical samples. From June 2021 to August 2022, 101 clinical samples positive in PCR for partial 16S rRNA gene analysis were subjected to both Sanger and ONT sequencing. Sanger sequences were edited and compared with deposited sequences in the NCBI database using BLAST, while ONT data were processed using EPI2ME Fastq 16S. The positivity rate (clinically relevant pathogen) was higher for ONT vs. Sanger sequencing: 72% and 59%, respectively. Concordance between Sanger and ONT sequencing was 80%. Furthermore, ONT detected more samples with polymicrobial presence compared to Sanger (13 vs. 5) sequencing. Interestingly, in one joint fluid sample, Borrelia bissettiiae was identified by ONT but not by Sanger. The results show that the detection of both monobacterial and multiple bacterial species is improved using ONT.