Basic reporting

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Experimental design

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Validity of the findings

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Comments for the author

This is a very useful protocol that will come in very handy for a large number of people. I look forward to having it available for undergraduate teaching in my own department. However it could benefit from some reorganization to make the workflow more clear. Because this is a protocol, side steps, optional steps, and parallel steps should be clearly designated. Also, the hierarchy of the organization system should be preserved across techniques.

Key suggestions:
1. Rooting the entire organizational structure around an improved workflow graphic would probably be the best place to start. I will make an attempt to include my own hasty sketch of this to make my comments more easily interpretable (See attached pdf. If nothing comes through, I'll provide upon request via email). The major sections of the workflow should correspond to major sections in the text.

2. Sections 2-4 might better be moved to the end as an appendix.

3. DNA extraction can be used in parallel for 16S analysis and genome sequencing, so this creates a set of two parallel processes from that point.

4. Make all the processing steps (RDP, SeqTrace, custom scripts) subsections of a larger whole (e.g., Sanger Sequence Processing)

5. Add a section on genome sequencing. Right now there is a section for Sanger sequencing, but nothing for the actual genome sequencing.

6. As a subset of assembly, add some comments about demultiplexing options, since multiplexing is discussed previously in some detail.

7. Demote section 10 to a subset of annotation

8. Break out section 11 as an analysis methodology that can be used in service of the first parallel chain (16S ID) and the second (whole genome sequencing).

9. Add a section on publication. There are several options, from genome announcements at J Bact, like you have done, to SIGS, Marine Genomics and PLOS. These have different appeals and drawbacks and make a logical conclusion to the steps herein.

10. The last paragraph of section 7.4 (lines 547-553) is a big issue and deserves to be extracted and expanded upon in the introduction or as an appendix item like the other “General Notes” items. Any considerations here that might help people select strains for sequencing that are of the most benefit to the community will add value to their efforts.

Minor comments
Section 2.2- change to “Summary of common commands and terms”
In the flow chart, “colony PCR” is used, but “direct PCR” is used in the text.
Section 5.2- include a reference for a plate making protocol.
Section 6, line 218- following “liquid culturing,” instead of “second dilution streaking”
Line 225, 257, 265, elsewhere, remove the reaction from “PCR reaction”- it’s redundant.
Line 240, “…use of a phenol and chloroform extraction…”
Line 387, 400, elsewhere, figure designations in the text are messed up
Add Figures 6-8 to the text somewhere. I didn’t see them mentioned.
Section 7.4 is really parenthetical to the regular blast-based approach mentioned earlier, since you will get many alignments to view. It might be worth adding/combining this to the processing steps.
Figures 7 and 8 need some kind of discussion in the legend about where the trees came from, and why there are being used, because as they are positioned in the text, the novice may wonder why they don’t get trees from their blast results.
Line 610- …multiplex (Section 8.10) to achieve…