Automated 16S Sequencing Using an R-Based Analysis Module for Bacterial Identification

Sanger sequencing of the 16S rRNA gene is routinely used for the identification of bacterial isolates. However, this method is still performed mostly in morespecialized reference laboratories, and traditional protocols can be labor intensive. In this study, 99 clinical bacterial isolates were used to validate a fast, simplified, and largely automated protocol for 16S sequencing. The workflow combines real-time PCR of the first 500 bp of the bacterial 16S rRNA gene and amplicon sequencing on an automated, cartridge-based sequence analyzer. Sequence analysis, NCBI BLAST search, and result interpretation were performed using an automated R-based script. The automated workflow and R analysis described here produced results equal to those of manual sequence analysis. Of the 96 sequences with adequate quality, 90 were concordantly identified to the genus (n = 62) or species level (n = 28) compared with routine laboratory identification of the organism. One organism identification was discordant, and 5 resulted in an inconclusive identification. For sequences that gave a valid result, the overall accuracy of identification to at least the genus level was 98.9%. This simplified sequencing protocol provides a standardized approach to clinical 16S sequencing, analysis, and quality control that would be suited to frontline clinical microbiology laboratories with minimal experience. IMPORTANCE Sanger sequencing of the 16S rRNA gene is widely used as a diagnostic tool for bacterial identification, especially in cases where routine diagnostic methods fail to provide an identification, for organisms that are difficult to culture, or from specimens where cultures remain negative. Our simplified protocol is tailored toward use in frontline laboratories with little to no experience with sequencing. It provides a highly automated workflow that can deliver fast results with little hands-on time. Implementing 16S sequencing in-house saves additional time that is otherwise required to send out isolates/specimens for identification to reference laboratories. This makes results available much faster to physicians who can in turn initiate or adjust patient treatment accordingly.

Automated summary

In this study, a fast, simplified, and largely automated protocol for 16S sequencing was validated using 99 clinical bacterial isolates. The protocol combines real-time PCR and automated amplicon sequencing, with sequence analysis and result interpretation performed using an automated R-based script. The protocol achieves a high accuracy rate of nearly 99% compared to routine laboratory identification methods. This simplified sequencing protocol is suitable for frontline clinical microbiology laboratories.