Open preprint reviews by Keith Robison

Improved de novo Genome Assembly: Linked-Read Sequencing Combined with Optical Mapping Produce a High Quality Mammalian Genome at Relatively Low Cost

David W Mohr, Ahmed Naguib, Neil Weisenfeld, Vijay Kumar, Preyas Shah, Deanna M Church, David Jaffe, Alan F Scott

Table 1 -- re-ordering the columns in a logical progression from left-to-right would scan better -- so DISCOVAR, 10X Supernova 1.1, BNG+Supernova 1.0, BNG+Supernova 1.1 (and a bit odd that Supernova 1.0 is omitted)

Table 2 takes a lot of space and isn't really giving more information than Figure 5 -- it would be preferable to have plots like Figure 5 for more chromosomes or a table listing the number of scaffolds & the sizes of scaffolds for each chromosome

The fact that the BNG data greatly reduced the number of scaffolds but had only a modest effect on N50 should be discussed. Is this a limit on scaffolding through centromeres? Do any scaffolds appear to cover an entire chromosome arm? Do any cross a centromere? It might be useful to discuss the known chromosome structure of pinnipeds as described in Beklemisheva 2016 -- adapting their Figure 4 to show how your scaffolds relate to human-seal and dog-seal synteny blocks would be valuable.

Figure 4 -- what region is this? Citation for the fact it is a breakpoint in many genome comparisons? Does this map to a known join vs. human karyotype as described in Beklemisheva? This point would be interesting to see discussed.

p.7 "acrocentric human chromosome which are" -- should identify which acrocentric chromosome(s) are being referred to.

It would be of interest to the genomics community to have a histogram of estimated fragment lengths based on the 10x read clouds and the observed lengths of BNG fragments. It would also be useful to have statistics on anomalously-mapping reads -- those that map outside the scaffold to which the majority of the cloud's reads are assigned. A histogram of number of reads per UMI might also be interesting.

Figure 1 -- move the legend into the plot by labeling the lines-- much easier to read, particularly for the colorblind (red vs. green is never a good choice for that reason)


Discovered a small hitch in one thing I suggested -- the Baikal seal in the Beklemisheva analysis has 2n=32 but Hawaiian monk seals have 2n=34 (Lu et al 2000). According to Arnason 1974 the 2n=34 karyotype is probably ancestral with a single fusion generating the 2n=32 karyotype. Fronicke et al 1997 would make the fused chromosome "S", which is homologous to human chromosomes 17 and 5. Some more musings over on the blog

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A portable system for metagenomic analyses using nanopore-based sequencer and laptop computers can realize rapid on-site determination of bacterial compositions

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

Figure 4b: please make ines much heavier & cinder avoiding color for distinguishing stages - different shapes would work better (e.g. rectangle, hexagon, oval) for individuals with color perception issues.

If you are going to claim portability, then a complete list of equipment is needed, with weights. What are power requirements? Refrigeration requirements?

What is the yield from each run? What fraction of reads in each were unclassifiable? What is expected sensitivity in a more complex sample?

Discussion and legend for 4b should emphasize that you started with purified DNA, not bacteria. So time to lyse&purify DBA would need to be added to running time, and differential lysis/extraction could shift your sensitivity.

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INC-Seq: Accurate single molecule reads using nanopore sequencing

Chenhao Li, Kern Rei Chng, Jia Hui Esther Boey, Hui Qi Amanda Ng, Andreas Wilm, Niranjan Nagarajan

Section 2.2, line 12 " pooled (by mass)" -- does "by mass" meaning normalized to equal masses of each input?

Table 1 -- Defined abundances have only single significant digit -- is this indicative of the precision of the mixing? The SMRT & ONT 2D are given to two significant figures -- is this appropriate? Table 3 gives the values to 3 significant figures .

Table 2 has 0 for the input abundance of the Klebsiella strains -- is this correct? If so, why was it detected in the community?

Are the methods appropriate to the aims of the study, are they well described, and are necessary controls included? If not, please specify what is required in your comments to the authors.

Yes.

Are the conclusions adequately supported by the data shown? If not, please explain in your comments to the authors.

Yes.

Does the manuscript adhere to the journal’s guidelines on minimum standards of reporting? If not, please specify what is required in your comments to the authors.

Yes.

Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review? If an additional statistical review is recommended, please specify what aspects require further assessment in your comments to the editors.

There are no statistics in the manuscript.

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 have been a participant, via my company, in the Oxford Nanopore MinION Access Program,
which has arguably provided reagents with greater value than the $1000 entrance fee.

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.

Authors' response to reviews: (https://static-content.springer.com/openpeerreview/art%3A10.1186%2Fs13742-016-0140-7/13742_2016_140_AuthorComment_V1.pdf)


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

Judith Risse, Marian Thomson, Garry Blakely, Georgios Koutsovoulos, Mark Blaxter, Mick Watson

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.


The reviewed version of the manuscript can be seen here:

All revised versions are also available:

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

Judith Risse, Marian Thomson, Garry Blakely, Georgios Koutsovoulos, Mark Blaxter, Mick Watson

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.


The reviewed version of the manuscript can be seen here:

All revised versions are also available:

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