PONE-D-19-18103

Screening of differentially expressed immune-related genes from spleen of broilers fed with probiotic Bacillus cereus PAS38 based on suppression subtractive hybridization

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The Authors aimed to construct the spleen differential genes library of broilers fed with probiotic Bacillus cereus PAS38 by suppression subtractive hybridization and screen the immune-related genes. The Authors have investigated an interesting topic and the theme has been properly described.

I would like to congratulate authors for the good-quality of the article, the literature reported used to write the paper, and for the clear and elegant and appropriate structure. The manuscript is well written, presented and discussed, and understandable to a specialist readership.

In general, the organization and the structure of the article are satisfactory and in agreement with the journal instructions for authors. The subject is adequate with the journal scope.

The work shows a conscientious study in which a very exhaustive discussion of the literature available has been carried out. The introduction provides sufficient background, and the other sections include results clearly presented and analyzed exhaustively.

As specific comment, I suggest to supply more high-quality figures.

Reviewer #2: This manuscript used suppression subtractive hybridization to identify genes that could have differential expression in response to the oral probiotic Bacillus cereus PAS38. They identified 119 valid ESTs and focused on 9 with immune related functions. Of these, only 3 were validated by qRT-PCR to have a treatment effect.

1. Why was SSH selected as the method for predicting differentially expressed genes? Since the chicken genome is fairly well annotated, why was RNA-seq or 3’ Quant-Seq (better suited to large sample numbers) not used? These methods can be statistically tested (unlike SSH) and also screen the whole transcriptome without a priori knowledge of the treatment effect.

2. Perhaps the low confirmation rate of the qRT-PCR reflects the lack of normalization to a reference gene. Even for absolute qRT-PCR, the copy number for each test gene needs to be normalized to a reference gene to account for variation in total input RNA. Please add the reference information if already done, or complete an additional qRT-PCR using a stably expressed reference gene, adjust the copy number for the 9 target genes and run the ANOVA again.

3. Please strengthen the evidence in the introduction for Bacillus cereus as a probiotic.

4. The discussion included very few references for chicken, with none for the significant JCHAIN, FTH1, and P2RX7 genes. Please add literature for these 3 genes in chicken.

5. The GO term results in lines 149-152 are not interpreted clearly. As analyzed, the biological process, molecular function and cell component terms are considered as if they are mutually exclusive and each EST can only have 1 type of term. It would be better to report this information as the percentage of the total ESTs in each category (so each EST can be represented 0-3 times) rather than a percentage of total GO terms.

6. Figure S6 and S8 show that IGF1R and ITGA4 primers have really low efficiency (60%). Were any other primer pairs tested for these genes? Perhaps an alternate would amplify better and better discriminate between the treated and control groups.

7. A gene set enrichment analysis (such as with PANTHER or GSEA) might be more informative than the top level GO terms from DAVID.

8. Based on the feeding protocol, there were 30 control and 30 treated birds. How many spleens from each group were used to perform the SSH and were the samples pooled? How many individual samples/group were used in the qRT-PCR validation? Add to the text and to figure legends. Also note that if all 60 birds were used, the phrase “randomly selected” in line 81 doesn’t make sense.

9. There are many other experimental details missing from the methods section, including:

a. What is the “basic diet”? Please move Table S3 into the main manuscript, as the composition of the diet is important to the interpretation of this study.

b. Please put the information in Table S1 and S2 into the text of the methods and not supplemental tables, so that the company and reagent information is complete in the text.

c. What type of housing (floor pens, cages, etc.) was used for the birds?

d. Please add the bacterial growth conditions (media, temp, etc.), and how the bacteria concentration was determined to line 72-74.

e. In line 87, “diluted 5 times” is not clear. Please clarify if this means a 5-fold (1:5) dilution or 5 serial dilutions to what final dilution factor.

f. How much input RNA was used for cDNA synthesis in line 87 and 93-94? How much cDNA was used as input for the qRT-PCR reactions in line 116?

g. In line 98, what were the hybridization conditions? Also add to this section, the enzyme and condition used for digestion, as mentioned in line 138.

h. For all reactions (cDNA synthesis in line 87 and 93-94, nested PCR in line 98, liquid-culture treated PCR in line 102-103, and qRT-PCR in line 116), the components of the reactions and the cycling conditions (i.e. the program) need to be described. For the qRT-PCR, please also include a description of how the standard curves were generated/used and reference the supplemental figures.

i. Were amplicons from the qRT-PCR confirmed by sequencing?

j. In line 110, what version of the DAVID database was used?

k. What were the positive and negative controls for the SSH and qRT-PCR validation?

l. The percentage agarose used for electrophoresis is not consistent. Is it 1.2% (line 90 and 128) or 1.5% (line 133 and 141)?

10. Other comments:

a. There are English grammar errors in the manuscript, please check.

b. In Figure 2, how was the subset of samples shown selected?

c. Figure 3 and 4 are blurry. Please improve image resolution.

d. In Table 2, clarify if up/down references treated vs control or control vs treated.

e. There are no references to the supplemental figures in the text. Please add.

f. In line 70 and 76, the birds are listed as “Avian broilers”. Was this supposed to reference an Aviagen broiler

line? If so, please correct. If “Avian” is just referring to bird, remove this word.

g. In line 41-42, reference 5 is cited for using appendix, which is not a tissue in birds. Are they referring to the ceca?

h. In line 109, what does “not matched” mean? No hit to chicken genome?

i. The number of valid ESTs are reported in line 147 but how many of the failed clones were low quality sequence, unmatched sequence, repeat sequence, or unannotated protein sequence?

j. ITGA4 and DOCK10 should not be highlighted in the abstract and conclusions as these were not confirmed by qRT-PCR (p>0.05).

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Reviewer #1: No

Reviewer #2: No

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Screening of differentially expressed immune-related genes from spleen of broilers fed with probiotic Bacillus cereus PAS38 based on suppression subtractive hybridization

PONE-D-19-18103R1

Dear Dr. PAN,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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Juan J Loor

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The Authors have revised their paper according to the comments received, so in my opinion the revised paper merits the final acceptance.

Reviewer #2: Thank you to the authors for their detailed work addressing the comments in their response letter and revised manuscript.

Two tiny comments:

In the new Blast2Go pie charts, there are watermarks behind the images. The images would be clearer without these.

As the corrected qPCR analysis now confirmed (p-value<0.05) the down-regulation of ITGA4 and DOCK10, these genes can be put back in the abstract and conclusions (contrary to the previous suggestion to remove them).

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No