A portable system for rapid bacterial composition analysis using a nanopore-based sequencer and laptop computer

Abstract

We developed a portable system for 16S rDNA analyses consisting of a nanopore technology-based sequencer, the MinION, and laptop computers, and assessed its potential ability to determine bacterial compositions rapidly. We tested our protocols using a mock bacterial community that contained equimolar 16S rDNA and a pleural effusion from a patient with empyema, for time effectiveness and accuracy. MinION sequencing targeting 16S rDNA detected all 20 of the bacterial species present in the mock bacterial community. Time course analysis indicated that the sequence data obtained during the first 5 minutes of sequencing (1,379 bacterial reads) were enough to detect all 20 bacteria in the mock sample and to determine species composition, consistent with results of those obtained from 4 hours of sequencing (24,202 reads). Additionally, using a clinical sample extracted from the empyema patient’s pleural effusion, we could identify major bacterial pathogens in that effusion using our rapid sequencing and analysis protocol. All results are comparable to conventional 16S rDNA sequencing results using an IonPGM sequencer. Our results suggest that rapid sequencing and bacterial composition determination are possible within 2 hours after obtaining a DNA sample.

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.…”

“…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.…”

Portable sequencer in the fight against infectious disease

“…Bacterial Fig. 1 The portable sequencer, MinION, is being handled prior to sequencing composition from empyema patient's pleural effusion could be understood within 2 hours after obtaining the DNA sample [29]. Here, the amplicons of 16s rRNA gene were sequenced to identify the bacteria.…”