The Clinical Utility of Two High-Throughput 16S rRNA Gene Sequencing Workflows for Taxonomic Assignment of Unidentifiable Bacterial Pathogens in Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming and low-throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests, which are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by respective built-in programs (MiSeq Reporter Software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to Sanger 16S and was better than Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78h and 8.25h respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

The clinical utility of Nanopore 16S rRNA gene sequencing for direct bacterial identification in normally sterile body fluids

“…The long-read Nanopore sequencing is advantageous for sequencing the entire 16S rRNA gene, which allows better taxonomic resolution at the species level. A previous study conducted by our group demonstrated that Nanopore sequencing achieved a diagnostic accuracy of 96.36%, which was similar to that of Sanger sequencing, for bacterial identification using 16S rRNA gene sequencing in clinical isolates (Lao et al, 2022). Due to its high sensitivity, real-time analysis platform, and the ability to identify mixed species, Nanopore 16S rRNA gene sequencing (Nanopore 16S) of direct specimens could be a potential alternative for rapid bacterial identification in clinical laboratories.…”

Alteration of gut microbiota in wild-borne long-tailed macaques after 1-year being housed in hygienic captivity

“…The full-length 16S sequencing workflow based on Oxford Nanopore Technology has the advantage of greater efficiency in species classification 15 . This strategy is also cost-effective 16 due to the cheaper reusable flow cells, more flexible input sample requirement and a rapid turnaround time 17 . At present, we investigate the alteration of the gut microbiome in wild-borne macaques that were translocated and housed in a hygienic facility for 1 year.…”

Application of Nanopore Sequencing in the Diagnosis and Treatment of Pulmonary Infections

“…The time taken by Illumina sequencing was 78 h, whereas nanopore sequencing required 8.25 h. Furthermore, the study found that although Illumina sequencing had high accuracy in base recognition, nanopore sequencing provided a higher taxonomic resolution at the species level. Therefore, compared with MALDI-TOF MS and Illumina sequencing, nanopore sequencing technology has a higher species identification ability, despite its higher sequencing error rate [ 75 ].…”