|The authors have made a sincere and detailed effort to improve the manuscript by addressing comments made in the earlier reviews. I am still wary about the use of measurements at 25*C with soils conditioned to 5*C, but can accept its necessity for the present study. The open nature of the review process offers readers with the inclination an opportunity to view the arguments for and against the use of 25*C temperatures.|
Sequencing 16S rRNA genes to parameterize the model. I still have problems here. It is assumed that shifts in the absolute quantities of different functional/taxonomic groups are meaningfully represented by DNA extracted from varying quantities of samples, amplified and sequenced to an unstated but incomplete depth, used to generate relative abundance profiles of sequence libraries which may differ in read number, and in any case are based upon sequences from a locus which has variable numbers of copies per bacterial genome, a source of variation which is known to be related to ecological traits important in glacier forefield successional processes and itself recapitulated within the data. Specifically:
A. L179: Thanks for updating the details here, but I have concerns over “5-10 grams of sample” being used for DNA extraction.
1. If this is the case, the statement DNA was extracted by “MoBio PowerSoil® DNA Isolation Kit and by following the instruction manual cannot be the case. This kit processes samples in 2 mL tubes for bead beating. The standard recommendation from the manufacturer is for 250 mg samples (and I have had no problems with getting enough DNA from forefield soils for amplicon sequencing from 250 mg using the standard protocol.) The key point is that 5-10 grams would not fit, and any more than 250-500 mg clogs the bead beating tube to the point that extraction efficiency is reduced, making using more sample counterproductive, so I think there’s a mistake here. Did the authors actually use a PowerMax kit which can process up to 10 g of sample in a very different format, or modify the powersoil protocol to pool extracts from multiple 250 mg extracts? This may seem a trivial detail, but establishing how you extracted the DNA is important to establish whether the sequencing data have any relation to the soil community.
2. So, some of your samples were 5 grams and some were 10 grams? How did you account for up to two fold variation in sample mass, with the implications for the total biomass in the tube, and furthermore the extractable biomass as a result of variation in extraction efficiency from the consequently variable ratios and mixing in volumes of sample, beads, buffer and tube during bead beating?
B. L193-200: To establish the depth of sequencing and how many reads were there per barcode, and was the depth of sequencing? In other words, how can the reader be assured that your sequencing effort meaningfully describes the community, and that your relative abundance profiles are from roughly equivalent (or rarefied) numbers of reads. This matters because you are using proportional data from sequencing libraries to make estimations of absolute contributions to the soil community your model describes.
C. From the taxonomic profiles I see important changes in the community’s composition and configuration which mirror other work on transitions between oligotrophic and copiotrophic taxa in a variety of glacier chronosequences. That transition is associated with changes in ribosomal RNA gene copy number per genome. There are bioinformatic means of normalising this variation in rRNA copy number, for example CopyRighter (DOI: 10.1186/2049-2618-2-11).
I would hope the authors can state succinctly the measures taken to address problems A and B, and caveat their discussion to address concern C. This would serve to improve this paper, and perhaps consideration of these issues will help the other paper mentioned in their response which will probably rely on this 16S data a central pillar. As a general thought, for future endeavours and considering the broad taxonomic resolution desired by the authors to inform their model, I feel there are more appropriate tools for this job. If DNA based analyses were needed, qPCR or FISH would have provided data more closely linked to microbial abundance. Otherwise, PLFA would have provided taxonomically-informed estimates of contributions to biomass.
L180: At this point it is not isolated rDNA; it will be community genomic DNA, from which you will specifically amplify bacterial 16S ribosomal RNA genes (rDNA itself is a common misnomer too, by the way: the gene product is rRNA, hence rRNA gene)
Accession numbers: Hopefully NCBI will have returned the numbers soon.
Response: “ Assuming that predation rates and dormancy are the same on all functional groups (as is defined in SHIMMER),” I advise caution in making that assumption as these traits are varied across the breadth of soil microbiota.