Hi All,

I have been trying to use IsoformSwitchAnalyzeR (1.6.0) to analyze my RNAseq data. I followed the tutorial but encountered some issue (code and error attached below). I was wondering if anyone could help me fix it? I also had a question about which gtf file should be used as isoformExonAnnoation? The original one I used for the very first stringTie run? Or the merged gtf file from stringTie --merge?

Thanks for your time and help!!

aSwitchList <- importRdata( + isoformCountMatrix = stringTieQuant$counts, + isoformRepExpression = stringTieQuant$abundance, + designMatrix = myDesign, + isoformExonAnnoation = "merged.annotated.gtf", + isoformNtFasta = "scaffolds.fasta", + showProgress = FALSE + )

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... 9520 ( 19.63%) isoforms were removed since they were not expressed in any samples.

Error in sample.int(length(x), size, replace, prob) : invalid first argument

In addition: Warning message:

In importRdata(isoformCountMatrix = stringTieQuant$counts, isoformRepExpression = stringTieQuant$abundance, : No CDS annotation was found in the GTF files meaning ORFs could not be annotated. (But ORFs can still be predicted with the analyzeORF() function)

aSwitchList

Error: object 'aSwitchList' not found