To whom it may concern, I've been having some problems with consistency in my limma results for genes that are found to have significant differential transcript abundance. In a given example, I may have 4 different groups (a, b, c, and d) in an array set of 12. >From here, I make a contrast matrix that has contrasts for a-b, a-c, and a-d. Eventually, I output an eBaye's corrected contrast fit and I use decideTests from there to find out what genes are differentially expressed. My misunderstanding is that when I take away an entire group (such as removing all d's) and redo all steps in the limma analysis, I find that I end up with a different set of genes after using decideTests. I am confused here, because I would not think that removing group 'd' from the analysis would have an effect on contrasts a-b and a-c. If anyone could even hint to me a reason as to why this is happening, it would be greatly appreciated. Thanks and regards, Joseph Skaf -- Joseph Skaf PhD student Department of Cell and Systems Biology University of Toronto 25 Willcocks Street Toronto, Ontario M5S 3B3 [[alternative HTML version deleted]]

Hi Joseph, Joseph Skaf wrote: > To whom it may concern, > > I've been having some problems with consistency in my limma results for > genes that are found to have significant differential transcript abundance. > > In a given example, I may have 4 different groups (a, b, c, and d) in an > array set of 12. > > From here, I make a contrast matrix that has contrasts for a-b, a-c, and > a-d. Eventually, I output an eBaye's corrected contrast fit and I use > decideTests from there to find out what genes are differentially expressed. > My misunderstanding is that when I take away an entire group (such as > removing all d's) and redo all steps in the limma analysis, I find that I > end up with a different set of genes after using decideTests. I am confused > here, because I would not think that removing group 'd' from the analysis > would have an effect on contrasts a-b and a-c. > > If anyone could even hint to me a reason as to why this is happening, it > would be greatly appreciated. It's because of how the denominator for your contrast is computed. The denominator is computed using the intra-group variance for all the groups in your study, not just the two groups being compared in the contrast. So if you remove one of the groups, you lose both degrees of freedom as well as the contribution from the intra-group variance of that group. Losing the degrees of freedom will reduce your power to detect differences. Losing the contribution of the intra-group variance will depend on how variable the group d data are compared to groups a-c. Best, Jim > > Thanks and regards, > Joseph Skaf > > > -- James W. MacDonald, M.S. Biostatistician Douglas Lab University of Michigan Department of Human Genetics 5912 Buhl 1241 E. Catherine St. Ann Arbor MI 48109-5618 734-615-7826 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues