Nanopore sequencing of the 16S rRNA (V1-V3) and rpoB genes directly from clinical samples.
Broad-range amplification and sequencing of the 16S rRNA gene enhances bacterial infection diagnosis. Recent improvements in sequencing accuracy of the Oxford Nanopore Technologies (ONT) sequencing platform, makes it a promising option for this approach in clinical microbiology. However, even full-length 16S rRNA sequencing may lack resolution to distinguish closely related species, a problem exacerbated by the remaining ONT error rate.  The rpoB gene is an alternative bacterial phylogenetic marker providing better species-level discrimination. We investigate a strategy combining ONT sequencing of the 16S rRNA (V1-V3) gene with a segment of the rpoB gene. Our results on complex clinical samples showed that rpoB ONT sequencing provided species-level identification in 91.2% of detections, as compared to 62.8% with 16S rRNA ONT sequencing. However, 16S rRNA ONT sequencing provided a higher sensitivity of 93.0%. Combining 16S rRNA and rpoB ONT sequencing yielded a sensitivity of 98.8% with 89.0% species-level discrimination. For both gene targets, ONT sequencing paralleled Illumina sequencing. Combining ONT sequencing with high-sensitivity broad-range amplification of the 16S rRNA gene and amplification of the rpoB gene for improved taxonomic resolution, enable accurate bacterial detection and identification directly from clinical samples. This approach represents a promising tool for clinical microbiology diagnostics.