Analysis of Faecal Microbiota and Small ncRNAs in Autism: Detection of miRNAs and piRNAs with Possible Implications in Host–Gut Microbiota Cross-Talk

Round 1

Reviewer 1 Report

Dear respected colleagues, I thank you for this impressive work where you addressed the presence of different types of sncRNA in ASD stool samples. 

I was looking for the conclusion that shows what did you find after all these experiments and analyses. Therefore, abstract and conclusion need representation to be more informative.  furthermore, in the introduction section, you missed some works that highlighted the role of gut microbiota in ASD which have been published recently. this is an example 

https://pubmed.ncbi.nlm.nih.gov/33553761/ 

meanwhile, some typo mistakes are there that need your attention. examples are

line 186: that instead of thet

line 215: revise 

line 221: displayed in 

Author Response

Dear Reviewer,

We thank you for revising the manuscript. We found your suggestions very useful to improve the quality of our manuscript and we revised the paper addressing the requests.

You: abstract and conclusion need representation to be more informative.

Authors: we improved abstract, discussion, and conclusion to make them more explanatory. You can find numerous revisions in the mentioned section of the manuscript.

You: you missed some works that highlighted the role of gut microbiota in ASD which have been published recently

Authors: we check the literature, and we found the paper you suggested very interesting, so we cited it (as ref 13) in the introduction and discussion sections.

You: typo mistakes

Authors: typos have been checked and rectified.

Best regards,

For the Authors

Reviewer 2 Report

The authors sequenced gut microbiota/mycobiota and small non-coding-RNAs from six patients with ASD and six controls. They tried to identify interactions among intestinal microbes and host transcriptional modulators in autism. Though interesting, the manuscript is not well organized, and the relationship between gut microbiota and host sncRNA is not fully presented and discussed. I suggest the authors concise the Result part. Below is my concerns.

Major concerns:

1. We usually call 16s/18s based study as ‘Microbiota’ instead of ‘Microbiome’, which is based on metagenome sequencing. So please change into microbiota throughout the manuscript.
2. In Part 3.1 and 3.2, statistical tests and p-values should be included. We’d like to see the difference between the two groups with statistical significance.
3. PiRNA in fecal samples had been reported, see mSystems 4:e00289-19. https://doi.org/10.1128/mSystems.00289-19.
4. The sequencing amount of 16s rDNA (ave 800,000) and 18s rDNA (ave 28,000) varied widely, this would affect analysis result. So rarefaction curve must be provided.

Minor concerns:

1. Line 97-98, need reference.
2. Line 163, 2.516. S？
3. Line 164, 18S rDNA, V3-V4? Line 216 is NS1-NS2.
4. Line 168, 50 million reads per sample? Please keep consistent with Line 216-217, it’s 0.8 million for 16s and 28000 for 18s.
5. Line 179, was reads normalized before OTU construction?
6. What does ASV and CSV mean in Figure 1?
7. Line 168, 2X300 base, 1x75bp?
8. Please spell out all the number less than 10.

Author Response

Dear Reviewer,

We thank you for revising the manuscript. We found your suggestions very helpful to improve the quality of our manuscript. We revised the paper addressing the requests.

We improved and re-organize discussion and conclusion sections to better discuss the effort in disentangling the host sncRNAs-microbiota interaction. We did not concise the results because all presented results are necessary to the discussion.

Major concerns:

You: we usually call 16s/18s based study as ‘Microbiota’ instead of ‘Microbiome’, which is based on metagenome sequencing. So please change into microbiota throughout the manuscript.

Authors: we checked the correct use of “microbiome” and “microbiota” in the manuscript.

You: In Part 3.1 and 3.2, statistical tests and p-values should be included. We’d like to see the difference between the two groups with statistical significance.

Authors: we specified, in the methods section (2.7 and 2.8), the statistical tests we applied to metataxonomic and RNA-seq analysis, and reported in new supplementary table all the p-value of the metataxonomic analysis. The FDRs of the sncRNA analysis are already reported in the Supplementary Table 3.

You: PiRNA in fecal samples had been reported, see mSystems 4:e00289-19. https://doi.org/10.1128/mSystems.00289-19.

Authors: We modified the manuscript. In the abstract section (lines 29-30), “…for the first time, we detected miRNAs and piRNAs in faecal samples of patients with autism”. In the introduction (line 184), “…We applied a bioinformatics approach to correlate gut bacteria and fungi composition with host miRNAs and piRNA expression, for the first time in stool samples from patients with ASD, and tried to highlight possible mechanisms of microbiota-host bidirectional cross-talk in ASD”. In conclusion section (lines 1042-1043), “…Furthermore, for the first time we profile miRNAs and piRNAs in ASD stool samples, which targeted pathways with roles in ASD”.

You: The sequencing amount of 16s rDNA (ave 800,000) and 18s rDNA (ave 28,000) varied widely, this would affect analysis result. So, rarefaction curve must be provided.

Authors: The sequencing amount of 16S and 18S are different, but we analyzed prokaryotes and eukaryotes independently. We calculated rarefaction based on 16S and 18S OTUs, and rarefaction curves indicated that our sampling is sufficient to estimate the diversity for both 16S and 18S, as you can see in the attached image.

Minor concerns:

You: Line 179, was reads normalized before OUT construction?

Authors: No, Variance stabilizing transformation was applied to normalized across samples on OUTs with DESeq2, as described by McMurdie and Holmes in Plos Computational Biology 2014. This has been added in the manuscript, lines 354-356.

All other minor concerns have been addressed.

Best regards,

For the Authors

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Most of my concerns are addressed. Now only a few problems existed.

1. In Figure1, ASV and CSV should be explained or modified into ASD and Ctrl? Same as in Figure 4.

2. At the end of Line 201, delete 3.

3. The raw data of 16s/18s and sRNA sequence must be deposited in a public database.

Author Response

Dear Reviewer,

We thank you for revising again the manuscript. We revised the paper addressing the requests:

- substitution of ASV and CSV with ASD and Ctrl also in Figures 1 and 4;

- deletion of “3” at line 201;

- we are depositing to SRA and GEO databases the raw data of 16s/18s and sRNA sequences. These procedures require up to five business days to be approved. Therefore, we will communicate to the editorial board the accession numbers provided by the databases and those will be included in the submitted manuscript as soon as possible.

With regard

For the Authors