Alternative Splicing detection and tools (Kallisto, sleuth, isoformSwitchAnalyzeR)

Question

[joomi]

Hello,

I hope this actually leads to a few answers, but I have RNASeq data and I want to study alternative splicing/isoform switch between two conditions.

I'm not sure what tools to use, but I have done transcript quantification with Kallisto. The results are on a cluster and overall we have 672 samples so it is impossible to move these directories into my personal computer.

My next step is to use Sleuth, however sleuth is in R. How do people use the cluster for quantifying and then use Studio for Sleuth?

Additionally, one other question I have is about using Sleuth for Alternative Splicing. I believe that Sleuth is not the best for Alternative Splicing and that I should use IsoformSwitchAnalyzeR instead. Which is also on R.

My second question mainly is: Can sleuth be used for Alternative Splicing or is it just for DGE?

Thank you!!

Answer 1

[Kristoffer Vitting-Seerup]

Sleuth is only for DE so you cannot use that to analyze splicing. You can use the Kallisto quantifications though :-)

I've written a section about the different approches to analyzing splicing in the vignette of my R package IsoformSwitchAnalyzeR.

[joomi]

Hello again,

I was wondering if I could ask about this error:

Step 1 of 6: Checking data... Step 2 of 6: Obtaining annotation... importing GTF (this may take a while) Step 3 of 6: Calculating gene expression and isoform fraction... 632 ( 0.85%) isoforms were removed since they were not expressed in any samples. Error in sample.int(length(x), size, replace, prob) : invalid first argument

I'm not sure what it means. I gave it this command: High_SwitchList<-importRdata(isoformCountMatrix=Kallisto_quant$counts,isoformRepExpression=Kallisto_quant$abundance,designMatrix=High,isoformExonAnnoation="/group/runciegrp/SharedResources/Genomes/Zea_mays/B73/AGPv5.0/Zm-B73-REFERENCE-NAM-5.0_Zm00001e.1.gtf",isoformNtFasta="/group/runciegrp/SharedResources/Genomes/Zea_mays/B73/AGPv5.0/Zm-B73-REFERENCE-NAM-5.0.fa.gz",showProgress=TRUE)