Question: Alternative splicing analysis following kallisto?
2
gravatar for dina.hesham139
14 months ago by
Egypt
dina.hesham139120 wrote:

Hi,

I used Kallisto for analyzing Rice RNAseq data, is there any tool that I can use to study alternative splicing following kallisto, a tool that doesn't require bam files and supports Rice genome?

Thanks in advance!

ADD COMMENTlink modified 14 months ago by kristoffer.vittingseerup1.9k • written 14 months ago by dina.hesham139120

Hey! So far you are the only one using this approach. I didnt find a single paper mapping RNAseq data against rice transcriptome. Thats pretty curious

ADD REPLYlink written 3 months ago by lessismore630
4
gravatar for kristoffer.vittingseerup
14 months ago by
European Union
kristoffer.vittingseerup1.9k wrote:

Hi Dina

Take a look at my R package IsoformSwitchAnalyzeR (you need version 1.1.07 or higher) - it directly supports Kallisto data and can analyze alternative splicing both individual genes as well as genome wide level. See examples for individual genes (isoform switch) here or for the global analysis see this part of the vignette.

And as Istvan suggest if you are interested in novel splicing you need a different analysis pipeline.

ADD COMMENTlink written 14 months ago by kristoffer.vittingseerup1.9k

Hi kristoffer, I 'm trying to analyze my kallisto data with IsoformSwitchAnalyzeR but I get this error when I use the importRdata function, I get this error:

> aSwitchList <- importRdata(
+   isoformCountMatrix   = mySwitchList$counts,
+   isoformRepExpression = mySwitchList$abundance,
+   designMatrix         = designMatrix,
+   isoformExonAnnoation = "/rice/Oryza_sativa.IRGSP-1.0.37.gtf",
+   showProgress = T)
Step 1 of 6: Checking data...
Step 2 of 6: Obtaining annotation...
    importing GTF (this may take a while)
converting annotated CDSs
Error in importRdata(isoformCountMatrix = mySwitchList$counts, isoformRepExpression = mySwitchList$abundance,  : 

The annotation and quantification (count/abundance matrix and isoform annotation) seems to be different (jacard similarity < 0.95). 
Either isforoms found in the annotation are not quantifed or vise versa. 
Specifically:
 42346 isoforms were quantified.
 98663 isoforms are annotated.
 Only 42346 overlap.

This combination cannot be analyzed since it will cause discrepencies between quantification and annotation thereby skewing all analysis.

Please make sure they belong together and try again. For more info see the FAQ in the vignette.

knowing that I use the ensembl gtf annotation file that used in kallisto analysis.

Any Help?!

thanks in advance

ADD REPLYlink modified 7 weeks ago • written 7 weeks ago by dina.hesham139120

Hi Dina

Sorry for not seeing this earlier. Did you solve this problem?

ADD REPLYlink written 16 days ago by kristoffer.vittingseerup1.9k
3
gravatar for Istvan Albert
14 months ago by
Istvan Albert ♦♦ 80k
University Park, USA
Istvan Albert ♦♦ 80k wrote:

Since with Kallisto you are quantifying against transcripts hence you can go ahead with your analysis.

If you trying to discover new splice variants you would need to align against the genome with a different method.

ADD COMMENTlink written 14 months ago by Istvan Albert ♦♦ 80k

Thanks alot Istvan ! What other pipeline would you recommend for studing alternative splicing? esp. for rice and in my case I have a condition that doesn't have a replicate (i.e. I can't use rMATS for example!)?

ADD REPLYlink written 14 months ago by dina.hesham139120
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