Hi,

I am hoping that someone might be able to give me some advice on adjusting or controlling for batch effects in RNA-Seq data using Ballgown?

I have got RNA-seq data for different batches of sequencing for the replicates of the sample. I followed new tuxedo pipeline for my preliminary analysis of the data and found that there were difference in total number of PE reads between the two batches of the sequencing (batch 1: 27 million reads; batch 2: 20 million reads)- therefore I think this difference could be due to batch effects within my dataset.

Is it possible to control for batch effects within Ballgown pipeline using the stattest function?

Any advice on this would be gratefully appreciated!

Many thanks,

Amy

Just because you have different library sizes doesn't mean that you have a batch effect. It's common to have different library sizes, and that will be accounted for during the analysis. You should do either MDS or PCA plots in order to see if there are any batch effects, however.

As for fitting a model with a batch effect (if needed), you can specify any model matrix you like. See the vignette (which you should have already read, BTW), particularly the Differential expression analysis section.