Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinION™ nanopore sequencer

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.

Rapid sequencing‐based diagnosis of infectious bacterial species from meningitis patients in Zambia

“…Advances in DNA sequencing technology have now enabled obtaining real‐time DNA sequencing data using the nanopore‐based sequencer MinION (Oxford Nanopore Technologies, Oxford, UK) . Recently, remarkable performances of the MinION system have been reported for rapid bacterial identification based on sequencing of full‐length 16S rRNA gene amplicons . Using this sequencer in tandem with two laptop computers, we have developed a portable and rapid bacterial composition analysis system for the on‐site diagnosis of infectious diseases .…”

“…The first challenge is that the quality of nanopore sequencing was found to be considerably lower, by about 85% for 1D sequencing than that obtained using more common next‐generation sequencers such as Ion PGM (Thermo Fisher Scientific, MA, USA). However, with the advantage of long‐read sequencing, MinION can detect bacterial species based on the full‐length 16S rRNA gene, which improves the accuracy of species detection . Moreover, nanopore sequencing technology is continuously being updated, resulting in improvements in both the quality and quantity of the output sequencing data.…”

“…With these improvements, we recently updated our rapid diagnosis system for bacterial infection. Using this system, we have conducted sequencing analyses of mock bacteria samples and aspiration pneumonia to evaluate the DNA preparation methods and identify a causative agent, respectively. We successfully identified bacterial species in both studies.…”

“…1 Recently, remarkable performances of the MinION system have been reported for rapid bacterial identification based on sequencing of full-length 16S rRNA gene amplicons. [2][3][4][5][6][7][8] Using this sequencer in tandem with two laptop computers, we have developed a portable and rapid bacterial composition analysis system for the on-site diagnosis of infectious diseases. [2][3][4] Although this portable system could successfully determine the bacterial composition by sequencing 16S rRNA genes, there are two major challenges that need to be overcome for improved utility: the quality of DNA sequencing and the speed of sequencing searches.…”

“…However, with the advantage of long-read sequencing, MinION can detect bacterial species based on the full-length 16S rRNA gene, which improves the accuracy of species detection. [2][3][4][5][6][7][8] Moreover, nanopore sequencing technology is continuously being updated, resulting in improvements in both the quality and quantity of the output sequencing data. Therefore, the newer versions of MinION flow cells with updated library preparation kits should help to improve the quality issue.…”