Hello,

We have RNA-seq as well as functional data of various kinds.

For some of the most important genes in our study, we quantified protein and transcript levels using wet lab assays to confirm the size and direction of effect of the results.

Our original data were from microarray studies, these data showed massive downregulation of interferon responsive genes in the microarray data; subsequently, we have confirmed this in several different ways.

After running DESeq2 on the same samples, the results are completely congruent with prior studies. However, further processing the data with ashr or ApeGLM, as recommended by Michael Love elsewhere on this site and in published articles, the lfc direction flips.

this particular experiment has many more genes that are down than up. my understanding is that this is likely to affect results generated by Ashr, but not ApeGLM.

However, in either case, running lfcShrink produces values contrary to a great deal of accumulated experience.

This is addressed in the 2018 manuscript for apeGLM, but i cannot find suggestion therein why ApeGLM would behave in such a way in this case (as distinct from ashr, which does make sense granted the characteristics of the datasets). Any thoughts on this?

the lfc direction flips

Do you mean the sign flips for all genes? I'm guessing this has to do with how the factors are coded.

Can you plot(ashr LFC, apeglm LFC)