Mansour et al. report on the generation of a novel transcriptome assembly for Porites astreoides from multiple life history stages. P. astreoides is a fascinating coral species in that it is the only scleractinian in the Caribbean that is maintaining population densities [1] in the face of massive region-wide declines in coral cover [2]. A transcriptome is already available for P. astreoides [3] http://www.bio.utexas.edu/research/matz_lab/matzlab/Data.html; though, Mansour et al.'s assembly has the potential to be more informative and useful since it encompasses multiple life history stages similar to the transcriptome developed for Acropora millepora [4], whereas the previous assembly used only adult tissue.

However, before this potential can be realized, substantial re-analysis is needed. First, the authors do not report any methods for removal of Symbiodinium transcripts from the assembly (e.g. [3]) or partitioning the assembly into host and symbiont transcriptomes, as was done for another recent transcriptome, Porites australensis [5]. P. astreoides hosts populations of intracellular dinoflagellate symbionts throughout its entire life cycle. Symbiodinium are eukaryotic, and so poly-A based selection will capture symbiont mRNA in addition to host mRNA. Second, the assembly statistics are concerning. The authors report a transcriptome with 800K transcripts. This is an order of magnitude more contigs than previously published transcriptomes (~40-50K contigs on average) and genomes (e.g. Acropora digitifera ~23,000 gene models), which suggests a poor quality assembly. I recommend attemping a re-assembly using other scleractinian coral transcriptomes/genomes as a guide, in addition to removing or partitioning host and Symbiodinium contigs. Additional concerns are outlined in the specific comments below.

Specific comments:

Abstract - Pg. 2, line 6 - This sentence is contradicted by the main background section below where it is stated that P. astreoides is relatively resistant to anthropogenic stressors. I recommend revising this sentence to align with the main body of the note. Other scleractinians are threatened, but P. astreoides is very robust [1].

Pg. 2, line 14-15: change 595 million bp to megabases (595 Mbp)

Pg. 2, line 16: 800k transcripts is an extremely large. Did you filter for PCR duplicates and lowcomplexity reads? What was the coverage of your assembly?

Pg. 2, line 22: No mention is made of how host and symbiont transcriptomes are separated. Can you please add this to the methods/results.

Data description

Pg. 3, line 9: suggest changing "their response to environmental change" to "coral response to environmental change" for clarity

Pg. 3, lines 8-16: P. astreoides is a fascinating coral and I'm thrilled you're using this species as a model. But I think the point of a background justification is to outline why your work will be useful for the field as a whole rather than why it is useful for your future projects specifically. There is another transcriptome already available for Porites astreoides. How is your assembly better? I suggest revising this paragraph to discuss the potential interest of P. astreoides in that it is tolerant to climate change and other anthropogenic stressors, which may indicate interesting modifications at the genetic level. And to describe how your assembly improves upon prior work (multiple life stages vs adult only).

Pg. 3, lines 18-20: You do not report on differential expression profiling in this note. Please revise this sentence to indicate that here, you're reporting on the transcriptome assembly only.

Pg. 4, lines 1-2: The previous paragraphs do not provide adequate support for this conclusion. See previous comments for revision suggestions.

Samples

Pg. 4, line 19: I suggest explaining "seasoned" settlement tiles or providing a citation for a more detailed explanation

Pg. 4, line 22: So only a single adult was used? Were larvae and recruits offspring of this adult? Or were they mixed individuals?

Sample preparation and sequencing

General comment: this section is very confusing as written given the diversity of sampling years, library preparation and sequencing methods. I suggest adding in a summary table to better explain the interactions between samples/methods. See supplementary material from Moya's A. millepora transcriptome as an example - http://onlinelibrary.wiley.com/doi/10.1111/j.1365- 294X.2012.05554.x/full

Pg. 5, line 5: how many families were represented in each sample?

Pg. 5, lines 8-10: please describe quality control thresholds (e.g. RIN>8).

Pg. 5, lines 12-15: below you state there are five total samples, but here you list eight accession identifiers? Please clarify.

Pg. 5, line 22: by fragments do you mean sequencing reads? If so, I suggest changing to "reads" throughout. Also, how many samples for each life history stage were sequenced? And for the larval samples, were these 20 individuals each? Or 20 individuals per sequenced sample?

Transcriptome assembly

General comments: Watch spacing throughout around reference brackets. Pg. 6, lines 9-12: Please list quality filtering thresholds used. It seems like a very small fraction of your reads were removed due to poor quality. This stage is critical for good assemblies as de novo assemblers are very sensitive to sequencing errors.

Pg. 6, line 14: low complexity reads and polyA reads should be removed prior to assembly. I also suggest removal of PCR duplicates. This may be part of the reason why you have such an abnormally large number of contigs.

Pg. 6, line 20: 1e-03 is a fairly liberal blast threshold. I recommend 1e-04 at least. In addition, once you've re-assembled, using Cnidarian references for blasting (e.g. A. digitifera, N. vectensis) should improve your % annotation.

Carly Kenkel

References 1. Green, D.H., Edmunds, P.J., and Carpenter, R.C. (2008). Increasing relative abundance of Porites astreoides on Caribbean reefs mediated by an overall decline in coral cover. Marine Ecology Progress Series 359, 1-10.

2. Gardner, T.A., Cote, I.M., Gill, J.A., Grant, A., and Watkinson, A.R. (2003). Long-term region-wide declines in Caribbean corals. Science 301, 958-960.

3. Kenkel, C., Meyer, E., and Matz, M. (2013). Gene expression under chronic heat stress in populations of the mustard hill coral (Porites astreoides) from different thermal environments. Molecular Ecology 22, 4322-4334.

4. Moya, A., Huisman, L., Ball, E.E., Hayward, D.C., Grasso, L.C., Chua, C.M., Woo, H.N., Gattuso, J.P., Foret, S., and Miller, D.J. (2012). Whole transcriptome analysis of the coral Acropora millepora reveals compex responses to CO2-driven acidification during the initiation of calcification. Molecular Ecology 21, 2440-2454.

5. Shinzato, C., Inoue, M., and Kusakabe, M. (2014). A snapshot of a coral "holobiont": A transcriptome assembly of the scleractinian coral, Porites, captures a wide variety of genes from both the host and symbiotic zooxanthellae. PLoS ONE 9, e85182.

Level of interest

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An article whose findings are important to those with closely related research interests.

Quality of written English

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Needs some language corrections before being published

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