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Referee comments: Referee 2

Posted by PLOS_ONE_Group on 07 Mar 2008 at 18:40 GMT

Referee 2's review:

Review of the original submission:
This MS describes DNA methylation and expression status of ALVEs and TVB genes in chickens. The major finding is strain specific differential CpG methylation at the ALVE regions. Interestingly, strain L63 with hypermethylation at these regions are resistant to MD tumors. The methodology is correct and the results are fine. There are some key issues that could be addressed to improve the manuscript.

Specific comments:
Abstract: the authors state that ALVE region 3 and 4 were significantly lower in L63 in all tissues except liver (P<0.0001). Where is the P value from? Based on Figure 3, it is hard to agree that there are any significant differences between the two strains. The authors mention about "Epigenetic inheritance of ALVE-region 2", but they have no data to support it.

Introduction: a more germane introduction would focus on the distribution of ALVEs in the chicken genome, as well as the different tumor patterns among different strains.
Method: it would be worth to show bisulfite cloning and sequencing as validation of their pyrosequencing results.

Results: it is more appropriate to show the data instead of using P value only. Which tests were done for the comparison of methylation between two strains at different loci?
In "Association analysis between mRNA expression levels and DNA methylation levels", it is more appropriate to give R values for all regions analyzed instead of ALVE region 3 only.

Discussion needs to be rewritten to make a clear discussion.

Review of the first revised manuscript:
The authors have answered this reviewer's criticisms. The information included in this manuscript will be useful for the readers in the field. Publication is recommended.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.