Abstract
Rapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION™ sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable time frame for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate‐limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinION™ sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.
Highlights
We evaluated the performance of primer sets targeting the near-full-length 16S ribosomal RNA (rRNA) gene
Rapid detection and identification of bacterial strains via direct PCR amplicon sequencing on Having demonstrated the efficacy of the barcoded primers for amplifying 16S rRNA genes directly from bacterial suspensions, we investigated whether the direct PCR method can impact MinIONTM sequencing results and the accuracy of strain identification
Bacterial cell suspensions of E. coli and S. aureus were used for preparing 16S rRNA gene amplicon libraries, and the samples were sequenced on MinIONTM for 5 min (Table 1 and Fig. 3)
Summary
To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. This study establishes a relatively simple workflow for rapid bacterial identification via MinIONTM sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings. Targeted amplification of specific regions of the 16S rRNA gene followed by next-generation sequencing (NGS) is a powerful strategy for identifying bacteria in a given sample. A 5-min data acquisition using MinIONTM and sequence annotation against our in-house genome database enabled the molecular detection of bacterial DNA in a reasonable time frame for diagnostic purposes. To accelerate the process of sample preparation, we adopted a direct PCR strategy to amplify the 16S rRNA gene from bacterial extracts without DNA purification. Amplification conditions for fast PCR using the KAPA2GTM polymerase were as follows: initial denaturation at 95 °C for 3 min, 25 cycles of 95 °C for 15 s, 55 °C for 15 s, 72 °C for 30 s, followed by a final extension at 72 °C for 1 min
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