Hi,
I want to use mfuzz for clustering of RNA-seq results.
The problem is that I can't find easy solution how to average biological replicates.
I assumed there is simple function for averaging replicates by conditions using info from phenoData for ExpressionSet or sample info in case of DESeqDataSet.
I use DESeq2 so I have DESeqDataSet object. I've prepared ExpressionSet object with standardised FPKM values for mfuzz but I've realised that I need to average value for each condition.
I know, I could use aggregate to average data in data frame but it is prone to error if used for object with many columns.

Thank you,
I've used limma's method and it works. However I have to change
assaytoexprs.Why have you used
ntf <- normTransform(dds)before averaging? I assumed that I'll standardize data inmfuzzusingstandardisefunction.Use whatever you feel is correct, I just put together an example for the sake of demonstration. It's a random example. Note that before standardization, you would still typically normalize and log2-transform your data.
ATpoint Thank you for directing my attention to normalization. I assumed that using FPKM is ok as it was mentioned at
Mfuzzpage. Do you think I can use FPKM?Or do you think that approach presented at sthda in "Normalization using DESeq2 (size factors)" is ok for clustering?
What you usually do is to normalize data first with respect to library size and composition (that is what the size factors do), then log2-transform and then Z-scale aka standardize. In DESeq2 the
vstfunction does the two first points plus some magic extra that is beneficial for downstream analysis so I would go with that. Alternatively, log2-transformed normalized counts work as well. That is whatnormTransformdoes.Thank you very much for this clear explanation. Since my last post I've read several posts about normalization but haven't found such clear information.
ATpoint Should I first standardise data and then average replicates?