Question: Analyzing RNA Seq splice variants using spike-ins?
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gravatar for jc.szamosi
3.5 years ago by
jc.szamosi50
Canada
jc.szamosi50 wrote:

I'm about to start a splice variant analysis in RNASeq, and I was wondering if anyone has done this using spike-in controls.

I see that ERCC normalization can't be combined with CuffDiff2, because CuffDiff2 has its own normalization, but I was wondering if anyone is using ERCC controls anyway just as a sanity check, and to look at things like minimum limit of detection.

I've also found SIRV spike ins that are specifically intended for use with splice variant analysis, and I was wondering if anyone has any experience with them, and if CuffDiff2 is an appropriate tool to use in that case.

Thanks!

rna-seq • 1.0k views
ADD COMMENTlink modified 2.9 years ago by Satyajeet Khare1.4k • written 3.5 years ago by jc.szamosi50

I was under an impression that type of normalization (spike in or no spike in) won't make much difference to the detection of alternative splicing events, since it is relative expression analysis of isoforms of the parent gene in two samples. Even the lab validation of relative isoform expression is generally done by normalization to the expression to the parent gene. Let know your experiences.

ADD REPLYlink modified 2.9 years ago • written 2.9 years ago by Satyajeet Khare1.4k
0
gravatar for nwon
2.9 years ago by
nwon30
New Zealand
nwon30 wrote:

Bump!

I am also interested to see experiences of other colleagues, ERCC is good for LOD, but also reveals and confirms what is known, it's not a sensitive as RT-qPCR.

Another spike in that just came live are sequins, there is a Nature Comm paper on this and a website to gain access to them too! Link to Sequins

ADD COMMENTlink written 2.9 years ago by nwon30
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