Comments on the manuscript "High throughput identification of novel conotoxins from the
Chinese tubular cone snail (Conus betulinus) by multi-transcriptome sequencing" by Peng et al.
(Manuscript GIGA-D-15-00011)

General comments:

The study investigated the diversity of conotoxins in the cone snail Conus betulinus via
transcriptome sequencing. The study is of interest because the genus Conus is a highly diverse
group (>600 species that are venomous and predatory) and the venom in individual snails is
known to contain a large number of bioactive ribosomally-synthesized peptides (conotoxins).
This chemical diversity is what makes Conus species interesting to researchers in various fields
(molecular evolutionary biology, drug discovery, etc.). With the advent of next generation
sequencing, researchers have begun to leverage the power of this sequencing technology to
characterize the extent of conopeptide diversity in each species. What makes this study more
informative than other similar studies is that it sequenced not just one or two transcriptomes but
transcriptomes from a variety of samples that enable the researchers to address interesting
questions that have not yet been investigated before, or at least not at the same scale. Venom
duct transcriptomes (normalized and non-normalized) from several specimens and from two
tissues (venom duct and venom bulb) of Conus betulinus were sequenced using the Illumina
platform, supplemented by Sanger sequencing of 11,000 ESTs. The combination of the data sets
has helped ensure data quality. The data sets have been deposited to the NCBI SRA database.

On whether this paper should be classified as a Data Note rather than full research article given
GIGASCIENCE guidelines, it can be argued that while multiple transcriptome data sets were
generated, the size of the output and the complexity of the analysis may not quite fall within the
"large scale data" scope that the editor may want to strictly enforce. However, if we go by the
classification of the published GIGASCIENCE articles and given the the authors' detailed
analysis of their data, this paper may qualify as a research article. The editor has the final say.
Regardless, I recommend acceptance of this manuscript for publication.

Specific comments:

The study reports the following major interesting findings:

- venom bulb transcriptome sequences and comparison with venom duct transcriptome
sequences from the same specimens. The study reports that the venom bulb does express
conopeptide genes, most of which are not expressed in the venom duct, although the
expressed genes are fewer and the expression levels are lower.

- the researchers' sequencing strategy (sequencing of both venom duct and venom bulb;
use of normalized and non-normalized transcriptomes; sequencing from multiple
specimens of the same species) enabled the discovery of what the authors claim as the
highest transcript-level conotoxin diversity in a Conus species (215 distinct peptides) (but
see below) and discovery of a large number (17) of new superfamilies, a significant
number considering that only 26 superfamilies are currently recognized.

- their finding also confirms variability of conopeptide gene expression among Conus
individuals (and the first for C. betulinus).

The following revisions are suggested:

- p. 5, line 145: "vermivorous species . . . have been overlooked". Note that venom duct
transcriptomes from at least two vermivorous Conus species (C. pulicarius and C. miles),
sequenced using NGS, have previously been reported

- Methods section, p. 14, line 399: the basis for the taxonomic identification of the
specimens was not mentioned (shell morphology or molecular marker or both?) It might
help if pictures of the specimens are provided, at least as supplementary material.

- specific collection site(s) were also not mentioned (all specimens were collected from the
same site or from different sites?)

- in the "Prediction and identification of conopeptides" section, the authors should mention
the minimum depth of coverage for a unigene to be regarded as a putatively "genuine"
transcript (as opposed to being a potential sequencing artifact)

- the authors claimed that the number of inferred peptides (215) is the highest number so
far described; Jin et al (Mol Cell Proteomics. 2013 Dec;12(12):3824-33. doi:
10.1074/mcp.M113.030353) reported that they found 662 putative conopeptide encoded
sequences from Conus miles transcriptomic data.

- in a number of places, there seem to be inappropriate choice of words (e.g., "sequencing
of the whole mRNA reservoir", p. 6 line 150; "huge differences ...", p7 line 194;
"reconfirmed into", p. 8 line 209; "high homology", p.9 line 264; etc.)

- pp 235-240: correlation of differential expression with growth stages was made without
due consideration given to the fact that no replication of samples was made. Since
expression of conopeptide genes is known to be highly variable across individuals, could
have the observations been due to chance?
- Methods section, p. 15, line 415: the cDNA library from the "medium sized" specimen
was normalized, but the normalization procedure was not described.

- Methods section, p. 15, lines 434-436: different assemblers were used (SOAPdenovoTrans
for the Normalized venom duct transciptome, Trinity for the other transcriptomes),
but the choice of assemblers for specific data sets was not explained. In addition, could
these assemblers have yielded different results if used on the same data sets?

- Tables 1 and 2 and Figure 8: it might be necessary to add, say, a footnote to indicate that
different de novo assemblers were used for the different transcriptomes to ensure a more
informed comparison of the statistics.

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:
Needs some language corrections before being published

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
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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?

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Authors' response to reviews: (http://www.gigasciencejournal.com/imedia/3630358422010547_comment.pdf)