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. 2015 Mar 26:4:12.
doi: 10.1186/s13742-015-0051-z. eCollection 2015.

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

Andy Kilianski  1 , Jamie L Haas  2 , Elizabeth J Corriveau  1 , Alvin T Liem  1 , Kristen L Willis  3 , Dana R Kadavy  2 , C Nicole Rosenzweig  1 , Samuel S Minot  2
Affiliations

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

Andy Kilianski et al. Gigascience. .

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.

Keywords: Amplicon; Escherichia coli; Long-read sequencing; MinION™; NGS; Nanopore; Oxford Nanopore; Pathogen detection; Poxvirus; Whole genome sequencing.

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Figures

Figure 1
Figure 1
Distribution of read alignments along the reference using BLASR. Each mapped read (only 40 reads are depicted here) is depicted by a blue bar. Percent identity to the reference is displayed by the tint, with lighter blue being more identical to the reference. A majority of the reads mapped here were approximately 70-75% identical to the reference. Many mapped reads span the entire length of the reference, represented on the x-axis, with no overhang.
Figure 2
Figure 2
Bases aligned for vaccinia-Lister sample sequencing run using BLASR. The number of aligned bases for the vaccinia-Lister dataset are represented by the bars, having been aligned to a reference database containing the complete collection of known targets of the PCR primers that were used.
See this image and copyright information in PMC

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