Question: FPM value and batch correction
0
gravatar for Emir
10 weeks ago by
Emir0
Emir0 wrote:

Dear all, I have a bulk-rna dataset which I have analyzed by DESEQ2 and I have batch corrected it using limma package and removeBatchEffect() function. I would like to calculate the FPM value for expression data through fpm() function of deseq2. I know it will use the raw data to calculate these value. I have created a heatmap from my batch corrected data for some genew and I wanted to also plot the fpm value for these genes but then due to the fact I mentioned, the activity of my genes from heatmap in each condition will not exactly correspond to their expression from FPM table. How one should deal with this problem? can I also correct the FPM data for batches? What would be the appropriate way?

Thanks a lot!

limma deseq2 • 102 views
ADD COMMENTlink modified 10 weeks ago by Michael Love26k • written 10 weeks ago by Emir0
Answer: FPM value and batch correction
0
gravatar for Gordon Smyth
10 weeks ago by
Gordon Smyth39k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth39k wrote:

You seem to be mixing a lot of things, and it is not clear how you're doing that.

If you have run removeBatchEffect() in limma, then presumably you must have already converted to log-CPM values. So there's no need to run FPM because FPM and CPM are the same thing. If you want to unlog the log-CPM values, just use 2^x where x is the output from removeBatchEffects.

ADD COMMENTlink modified 10 weeks ago • written 10 weeks ago by Gordon Smyth39k

Dear Gordon, Thanks for you reply. Please have a look at my comments below and let me know if I am more clear now.

ADD REPLYlink written 10 weeks ago by Emir0

Dear Gordon, Thanks for you reply. Please have a look at my comments below and let me know if I am more clear now.

ADD REPLYlink written 10 weeks ago by Emir0
Answer: FPM value and batch correction
0
gravatar for Michael Love
10 weeks ago by
Michael Love26k
United States
Michael Love26k wrote:

Agree with Gordon that you are mixing various pipelines. As in the DESeq2 vignette if you want to remove batch effect variation for heatmaps etc, we recommend to do so on log scale data, so using vst() first. See the FAQ in our vignette.

ADD COMMENTlink written 10 weeks ago by Michael Love26k

Dear Michael and Gordon, Thanks a lot for your answers. Sorry if I was not that clear but yes for batch correcting my data I have used the log scaled data using vst and using this data, I have made heatmaps but I also thought it would be nice to have the count of genes as another plot beside my heatmap, that's why I wanted to use FPM or another normalized method such as RPKM. so I was thinking would be also a way to account for batches in that plot as it is calculated from raw data or what would be the best way to visualize these two data and show their relation.

ADD REPLYlink written 10 weeks ago by Emir0
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