On 2014 Dec 03, MARY PATTI commented:

Thank you very much for your interest in our study. We agree that genetic variation is an important consideration when conducting comparative epigenetic studies. However, we would like to clarify that we used samples of sperm from a total of 8 control (C) and 8 undernutrition (UN) mice for MeDIP-seq analysis. We prepared 2 pools for each condition, each comprising 4 individuals from 4 independent litters. These pools were used for 2 independent analyses (C pool 1 vs. UN pool 1, and C pool 2 vs. UN pool 2); differentially methylated loci common to both analyses were identified comprising clusters of CGs behaving similarly, not single site differences. Subsequent validation of these loci was very important. Pyrosequencing validation of differential methylation was performed on unpooled sperm using 12 individual control males from 5 litters and 11 individual UN males from 4 litters. This approach minimized outcomes potentially linked to inter-individual genetic differences.

On 2014 Oct 17, Andrew Sharp commented:

Readers should note that the whole genome-profiling in this study used outbred mice, with a sample size of n=4 per group. In my opinion, this is a very serious limitation, as it will result in the identification of many differences between groups that are simply due to the underlying genetic variation of the mice being tested, and are unrelated to nutrition status.

On 2014 Oct 17, Andrew Sharp commented:

Readers should note that the whole genome-profiling in this study used outbred mice, with a sample size of n=4 per group. In my opinion, this is a very serious limitation, as it will result in the identification of many differences between groups that are simply due to the underlying genetic variation of the mice being tested, and are unrelated to nutrition status.

On 2014 Dec 03, MARY PATTI commented:

Thank you very much for your interest in our study. We agree that genetic variation is an important consideration when conducting comparative epigenetic studies. However, we would like to clarify that we used samples of sperm from a total of 8 control (C) and 8 undernutrition (UN) mice for MeDIP-seq analysis. We prepared 2 pools for each condition, each comprising 4 individuals from 4 independent litters. These pools were used for 2 independent analyses (C pool 1 vs. UN pool 1, and C pool 2 vs. UN pool 2); differentially methylated loci common to both analyses were identified comprising clusters of CGs behaving similarly, not single site differences. Subsequent validation of these loci was very important. Pyrosequencing validation of differential methylation was performed on unpooled sperm using 12 individual control males from 5 litters and 11 individual UN males from 4 litters. This approach minimized outcomes potentially linked to inter-individual genetic differences.