Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer

Andy Kilianski¹^*, Jamie L Haas³, Elizabeth J Corriveau¹, Alvin T Liem¹, Kristen L Willis¹²,Dana R Kadavy³, C Nicole Rosenzweig¹ and Samuel S Minot³^*

*Corresponding authors: Andy Kilianski andrew.kilianski.ctr@mail.mil - Samuel S Minot sminot@signaturescience.com

Author Affiliations

¹Edgewood Chemical Biological Center, 5183 Black Hawk Rd Bldg E3150 Rm 324, Aberdeen Proving Ground 21010, MD, USA

²Defense Threat Reduction Agency, 8725 John J Kingman Rd Stop 6201, Ft. Belvoir 22060-6201, VA, USA

³Signature Science, LLC, 8329 N. MoPac Expressway, Austin 78759, TX, USA

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GigaScience 2015, 4:12 doi:10.1186/s13742-015-0051-z

The electronic version of this article is the complete one and can be found online at:http://www.gigasciencejournal.com/content/4/1/12

Received:	24 December 2014
Accepted:	27 February 2015
Published:	26 March 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Abstract

Background

The MinION™ nanopore sequencer was recently released to a community of alpha-testers for evaluation using a variety of sequencing applications. Recent reports have tested the ability of the MinION™ to act as a whole genome sequencer and have demonstrated that nanopore sequencing has tremendous potential utility. However, the current nanopore technology still has limitations with respect to error-rate, and this is problematic when attempting to assemble whole genomes without secondary rounds of sequencing to correct errors. In this study, we tested the ability of the MinION™ nanopore sequencer to accurately identify and differentiate bacterial and viral samples via directed sequencing of characteristic genes shared broadly across a target clade.

Results

Using a 6 hour sequencing run time, sufficient data were generated to identify an E. coli sample down to the species level from 16S rDNA amplicons. Three poxviruses (cowpox, vaccinia-MVA, and vaccinia-Lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains. The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%.

Conclusions

While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology. This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.