Hi,

I am using R version 4.3.1, R studio version 2023.6.1.524, isoformSwitchAnalyzeR version 2.0.1. I imported Salmon dataset via Tximeta and saved as RData. I created aSwitchList using importRdata(). I realized that 6 columns of SV values were created in the design matrix of aSwitchList, and these 6 columns caused error in running isoformSwitchAnalysisPart1(). I then removed them and successfully ran isoformSwitchAnalysisPart1(). But, I am curious what are those SV values and is it alright to manually remove them?

# Contents of sampletable_transcript_simple
>head(sampletable_transcript_simple)
   sampleID    condition
1  PFASdil1      GenX.KO
2  PFASdil4   Control.KO
3  PFASdil7      GenX.WT
4  PFASdil9      GenX.WT
5 PFASdil10  PFOA_0.3.WT
6 PFASdil13 PFOA_0.05.WT
> 


#Create data frame of comparisons
condition_table <- data.frame(condition_1 = c("GenX.WT", "GenX.KO", "PFOA_0.3.WT", "PFOA_0.3.KO", "GenX.WT", "GenX.KO"),
            condition_2 = c("Control.WT", "Control.KO", "Control.WT", "Control.KO", "PFOA_0.3.WT", "PFOA_0.3.KO")
                                )

#Create aSwitchList 
aSwitchList <- importRdata(
    isoformCountMatrix   = txi.transcripts$counts,
    isoformRepExpression = txi.transcripts$abundance,
    designMatrix         = sampletable_transcript_simple,
    isoformExonAnnoation = "gencode.vM32.primary_assembly.annotation.gtf.gz",
    isoformNtFasta       = "gencode.vM32.transcripts.fa.gz",
    comparisonsToMake = condition_table,
    fixStringTieAnnotationProblem = TRUE,
    showProgress = FALSE
)


#results
>head(aSwitchList$designMatrix)
   sampleID   condition         sv1         sv2         sv3         sv4
1  PFASdil1     GenX.KO  0.15325191  0.24239127 -0.08131793 -0.10151465
2  PFASdil4  Control.KO  0.08471039  0.03236587 -0.06652344 -0.39165379
3  PFASdil7     GenX.WT -0.01521535 -0.02794922 -0.42179622  0.19699090
4  PFASdil9     GenX.WT  0.20620929 -0.05459255 -0.03070380  0.08931218
5 PFASdil10 PFOA_0.3.WT  0.05922495 -0.04698004 -0.03901371 -0.11490429
7 PFASdil16 PFOA_0.3.KO  0.23170368 -0.04637444 -0.04477648  0.11710328
          sv5         sv6
1  0.04056574  0.16095426
2  0.20737072  0.29886258
3  0.64551612  0.04753523
4 -0.02190115 -0.04855234
5 -0.14909152 -0.49669229
7 -0.02094741 -0.03449496
> 

#Run isoformSwitchAnalysisPart1
SwitchList_p1.1 <- isoformSwitchAnalysisPart1(
                                          switchAnalyzeRlist   = aSwitchList,
                                          outputSequences      = TRUE,
                                          prepareForWebServers = FALSE
                                           )

#Error
>Step 1 of 3 : Detecting isoform switches...
Error: BiocParallel errors
  1 remote errors, element index: 1
  0 unevaluated and other errors
  first remote error:
Error in estimateDispersionsGeneEst(x, maxit = maxit, quiet = quiet, modelMatrix = modelMatrix, : the number of samples and the number of model coefficients are equal,
  i.e., there are no replicates to estimate the dispersion.
  use an alternate design formula
>

Thanks for reporting this problem.

The sv's added are the surrogate variables found by running SVA - meaning factors of unwanted variations. You might want to look into this, as they could repressent batch effects etc.

Cheers Kristoffer