Review for "A single chromosome assembly of Bacteroides fragilis strain BE1 from Illumina and MinION nanopore sequencing data"

Completed on 31 Aug 2015 by Keith Robison .

License: http://creativecommons.org/licenses/by/4.0/

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Comments to author

By demonstrating a closed bacterial genome generated by an Oxford Nanopore-Illumina hybrid assembly, the authors demonstrate a useful milestone. However, the presentation has a number of glaring flaws (as well as some smaller ones) which greatly decrease the potential value and impact of this manuscript. To start with one of the minor sins, the authors describe the competing PacBio sequencing as "uses a modified DNA polymerase and produces long...". This description of the technology relies solely on a factor which is neither unique nor informative: every polymerase-based high throughput sequencing system (and indeed vast amounts of Sanger data) have used modified polymerases. Given the amazing technology of the PacBio -- it, after all, uses optical methods to observe the incorporation kinetics of individual DNA polymerase enzymes -- this isn't a good start, since PacBio is the reigning leader in what the manuscript is doing. Of note, my office stapler is much larger than a MinION. Giving actual dimensions might be helpful. The authors mention the prior, all-nanopore, MinION assembly of a similar bacterium and note the error statistics for this assembly, setting up a comparison which unfortunately they fail to follow through with. While a number of figures and paragraphs are spent analyzing the accuracy of the Nanopore reads, only a scant paragraph on final assembly quality exists and this contains no statistics on the assembly. The authors appear to believe that all discrepancies between their assembly and the best available reference, a different strain of the same organism, are true strain differences, but the number of deviations are not specified. It would be also useful to understand the range of Illumina coverage across the final assembly, and in particular how well supported are the final joins and gap-fills made during the assembly process. It would also be of interest to many readers to understand better how much the MinION data boosted the assembly; comparison of a SPADES assembly made only with the Illumina data would be most instructive, even if only to give the number of contigs and the NG50, but actually analyzing the nature of what is successfully spanned by the MinION reads would be informative. It would be also useful for more analysis of why the initial MinION reads did not lead to a fullyclosed assembly. Analysis of the scaffolding process in this sort of detail -- what couldn't be spanned and why were other programs using the same data able to push through -- would raise this from a routine genome announcement to a useful addition to the genome assembly literature. Also, it is curious that the authors shade the all-MinION assembly with "However the assembly process was complex"; that paper used an error correction step, assembly and several rounds of polishing. This manuscript uses read trimming (of the Illumina reads), assembly, 2 rounds of scaffolding followed by one round of gap filling. It is difficult to see a significant difference in the level of skill required to implement either of these procedures. One other minor note, the mention of invertible promotes in the Discussion lacks the citation for this -- yes, it was used previously in the text, but since many readers may first jump in and encounter this item in this space, it is worth repeating the footnotes here. Level of interest Please indicate how interesting you found the manuscript: An article whose findings are important to those with closely related research interests Quality of written English Please indicate the quality of language in the manuscript: Acceptable Declaration of competing interests Please complete a declaration of competing interests, considering the following questions: 1. Have you in the past five years received reimbursements, fees, funding, or salary from an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? 2. Do you hold any stocks or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? 3. Do you hold or are you currently applying for any patents relating to the content of the manuscript? 4. Have you received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript? 5. Do you have any other financial competing interests? 6. Do you have any non-financial competing interests in relation to this paper? If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If your reply is yes to any, please give details below. I am, via my employer, also a participant in the Oxford Nanopore MinION Access Program, and as such receive free consumables for the system. However, I feel I can remain an impartial reviewer of data from this system, as I think this review will demonstrate. I agree to the open peer review policy of the journal. I understand that my name will be included on my report to the authors and, if the manuscript is accepted for publication, my named report including any attachments I upload will be posted on the website along with the authors' responses. I agree for my report to be made available under an Open Access Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any comments which I do not wish to be included in my named report can be included as confidential comments to the editors, which will not be published. I agree to the open peer review policy of the journal.


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