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Abstract

Background: The MinION (TM) 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 (TM) 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 (TM) 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 (TM) was accomplished despite an observed per-base error rate of approximately 30%.Conclusions: While nanopore sequencing, using the MinION (TM) 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 (TM) technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

Authors

Kilianski, Andy;  Haas, Jamie L.;  Corriveau, Elizabeth J.;  Liem, Alvin T.;  Willis, Kristen L.;  Kadavy, Dana R.;  Rosenzweig, C. Nicole;  Minot, Samuel S.

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  • 2 reviewers
  • The authors present a much improved manuscript with my major compulsory revisions met.

    Minor Essential Revisions

    1. Misspelling MinION as MinIO in introduction
    2. Line 127 – Sentence doesn’t make sense. “ The MinION reads generally represent to the complete length of the ...“
    3. Line 189 Missing a “0”? of .51 to 2.04

    Level of interest An article whose findings are important to those with closely related research interests Quality of written English Acceptable Statistical review No, the manuscript does not need to be seen by a statistician. Declaration of competing interests I declare that I have no competing interests

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  • Kilianski address the utility of the Oxford Nanopore MinION instrument in species/strain-level identification from three viruses and a bacterial strain through targeted amplicon sequencing. This is a novel and unreported experiment which will be of interest to those wishing to evaluate the MinION for clinical or environmental identification of microbes.

    My main issues with this manuscript concern the superficial level of reporting on the MinION sequencing. Given the pace of change in the MinION access programme there have been numerous chemistry changes (R6, R7, R7.3) and library preparation kit changes (SQK-MAP-001 to 004) as well as numerous protocol changes. Judging from the methods description this is likely to be R7 chemistry and SQK-MAP-002 with an additional overnight incubation stage. It is known by the MAP community that the overnight incubation step dramatically reduces yields, which might be one of the reasons for the very low numbers of reads retrieved during this study.

    Major Compulsory Revisions

    1) Something has gone wrong with these sequencing runs. It may be a fault of the library construction or perhaps supplied reagents. The generation of 296-1335 reads in a 6 hour run is well below expected performance for this device. The authors need to place these results in context with other reports and determine why the performance is so low (bad flowcells? overnight incubation step? other?). Did the authors do a lambda control library to determine whether these results are a result of library preparation? What QC values did these flowcells give? Ideally this would be repeated with latest chemistry (R7.3 and SQK-MAP-004) but if not possible then some additional context is needed here. 2) Improve reporting - what version chemistry, what version library kit, what METRICHOR workflow, what protocol version used? Any deviations from protocols? 3) Are the reads used one-direction (template and/or complement) or 2D? What is the % 2D? 4) Are they categorised as 'normal quality' (# template events > # complement events) or HQ2D (# complement events > #template events) - see Loman 2014 GigaScience for further definition. What % of each? 5) Why did the authors use BLASR for alignment? We found that for R7 normal quality 2D reads that LAST (or BWA MEM -ont2d) gives better results. 6) Was E. coli CS used in library preparation? 7) Where do reads not mapping to target organism come from? 8) "When analyzing the entire genome of E. coli (5-6mb) or poxviruses (150-280kb), the limited data generated after only 6 hours of runtime would make it difficult to identify or characterize organisms because of low coverage depth over a particular stretch of nucleic acids" - we were able to identify E. coli in a matter of minutes using our whole-genome datasets.

    Minor essential revisions

    9) Hanging sentence "see." in text.

    Discretionary revisions

    10) Figure 1 heat map is hard to interpret, could the % identities be reported in text?

    Level of interest An article of importance in its field Quality of written English Acceptable Statistical review No, the manuscript does not need to be seen by a statistician. Declaration of competing interests I am a participant in the MinION early access programme.

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  • Major Revisions

    1. The analysis of this paper relies on alignments generated with BLASR. They found that only a minority reads aligned to the known amplicon reference (10-20%). It has been shown that BLASR is not appropriate alignment method for minion data with LASTZ increasing the number of aligned reads of 1D data by ~50X and 2D data by ~ 10X (http://www.homolog.us/blogs/blog/2014/09/09/loman-and-browns-nanopore-presentation-live-feed/). As the authors analysis relies on the total number of bases aligned to each reference this is likely to be underpowered with this method. They should repeat the analysis using the appropiate tool.

    2. The metric of sum of length of all alignments needs to be explored with another analysis. I would like to see also an attempt to derive a consensus, or at least average identity to each reference.

    Level of interest An article whose findings are important to those with closely related research interests Quality of written English Acceptable Statistical review No, the manuscript does not need to be seen by a statistician. Declaration of competing interests I declare that I have no competing interest

    Published in
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