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

Satomi Mitsuhashi,Kirill Kryukov,Tadashi Imanishi,So Nakagawa,Junko S Takeuchi,Koichiro Asano,Yoshiki Shiraishi

doi:10.1038/s41598-017-05772-5

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.

Highlights

Time is of critical importance when identifying pathogens in acute infectious disease
The mock bacterial community contained bacterial genomic DNA at a ratio of equal 16S rDNA copy numbers for each species that we could obtain from the Biodefense and Emerging Infectious Research (BEI) Resources as a mixture[5]
Rapid 1D sequencing using SQK-RAD001 (Oxford Nanopore Technologies, Oxford, UK) is designed for fast library preparation, and utilises transposase to fragment DNA, simultaneously attaching the necessary sequencing adapters to its free ends. This is unsuitable for short amplicons; we amplified nearly full-length 16S rDNA (1,399 bp) for MinION Rapid 1D sequencing (Fig. 1b, Supplemental Tables 2 and 3)[6]

Summary

Introduction

Time is of critical importance when identifying pathogens in acute infectious disease. MinION’s rapid library preparation protocol was released in May 2016, which enables 10-min library preparation from DNA to bacterial identification This protocol only reads the template strand of double-stranded DNA (1D sequencing), and was initially considered to be of relatively low quality. Centrifuge is a novel microbial classification software that enables rapid and accurate identification of species, and can even be run on laptop computers[4] It is not clear what combination of these novel technologies is necessary to provide a sufficiently quick and reliable bacterial detection system in a clinical setting. We show that the combination of rapid sequencing using transposase-mediated library preparation for nearly full-length 16S rDNA amplicons, rapid 1D sequencing using R9 chemistry, 5-min sequencing data acquisition, local base-calling, and rapid analysis using Centrifuge on a laptop computer enables us to determine major bacterial lineages within 2 hours, even in a small laboratory environment

Methods

Results

Conclusion