DESEQ2 for circRNA normalisation
1
0
Entering edit mode
zoe.ward • 0
@zoeward-16214
Last seen 4.3 years ago

I’m a PhD student struggling to get my head around what I do to normalise my circRNA data (have tried to look at all of the related posts on various sites)

I am using DESEQ2 for differential analysis and have my gene matrix counts for the linear genes:

[1] dds <- DESeqDataSetFromTximport(txi, design_matrix, ~pair + cond)
[2] dds<- estimateSizeFactors(dds)
[3] dds<- DESeq(dds,parallel=TRUE)
[4] res.gene<- results(dds)

I now have a separate raw count matrix for the backsplice junction reads for my circRNAs.

To normalise would you suggest either of the following or something different?

Before line 2 (above) concatenate the circ raw counts matrix to the linear raw counts matrix for all samples and then proceed

Or

Somehow extract the SizeFactors and apply them to the circRNA count matrix? (If so how would I extract the size factors?)

Or Something different?

You advice would be GREATLY appreciated!

Zoe.

DESEQ2 circRNA normalisation • 1.4k views
ADD COMMENT
0
Entering edit mode
@mikelove
Last seen 6 hours ago
United States

I'm missing the biological question of interest. E.g. "I want to find genes in which ... is larger than ..., controlling for ..."

ADD COMMENT
0
Entering edit mode

Hi Michael, I have data from a group of patients measured at two time points (paired analysis). I want to identify circs that are differentially expressed between the two time points i.e I want to find circRNAs in which expression at time point B is larger/smaller than time A controlling for library size.

My understanding is that when analysing circs you can't normalise for library size on the total circRNAs alone.

ADD REPLY
0
Entering edit mode

Ok so I've got the DE between time points aspect. I just don't know what's the state of the art for normalization. So instead of looking across circRNA, you want to estimate size factors on the standard counts matrix?

That's easily done:

dds1 <- estimateSizeFactors(dds1)
sizeFactors(dds2) <- sizeFactors(dds1)
ADD REPLY
0
Entering edit mode

Thanks Michael, I'm getting this error though? Error in (function (classes, fdef, mtable) : unable to find an inherited method for function ‘sizeFactors<-’ for signature ‘"DESeqDataSet", "NULL"’

ADD REPLY
0
Entering edit mode

Just to be clear, you'll need to define the dds1 and dds2 above to be relevant to your dataset.

The code example is if you wanted to apply the size factors from one dataset to another. So I'm just showing how DESeq2 can be used. I don't happen to know the optimal normalization for circRNA, so you'll have to ask others for best practices. Here I provide software support.

ADD REPLY
0
Entering edit mode

Hi Michael,

I have used tximport to get my deseq object. Am I still able to extract the library size using sizeFactors()???

ADD REPLY
0
Entering edit mode

Not exactly, this has come up on the support site before, so see if you can find previous threads. Short answer is that you can get normalizationFactors() instead.

ADD REPLY

Login before adding your answer.

Traffic: 729 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6