Hi, I’m testing the rnasplice pipeline with some samples and I ran into this error using the

--edger_exon

option:

Loading required package: limma
  Error in eval(ej, envir = levelsenv) : object 'RNA_H2087_Rep1' not found
  Calls: makeContrasts -> eval -> eval
  Execution halted

I checked the file names and it looks like the error might be in the featureCounts.txt (needed for edgeR) because it has the name ‘RNA_H2087_Rep1_sorted.bam’ instead of ‘RNA_H2087_Rep1’ (and the names of the input files are correct).

# Program:featureCounts v2.0.1; Command:"featureCounts" "-F" "SAF" "-t" "exon" "-g" "gene_id" "-f" "-O" "-B" "-C" "-p" "-T" "6" "-a" "annotation.saf" "-s" "0" "-o" "RNA_H2087_Rep1.featureCounts.txt" "RNA_H2087_Rep1_sorted.bam" 
Geneid  Chr     Start   End     Strand  Length  RNA_H2087_Rep1_sorted.bam
ENSG00000000003 chrX    100627108       100627108       -       1       3
ENSG00000000003 chrX    100627109       100629986       -       2878    1459

The pipeline does this automatically, so any ideas on how to fix it? Thanks!

I don't really know what exactly is happening, but a colleague of mine had a similar error, and I believe it was the result of an error in the sample sheet or the contrast sheet, or a mismatch between them, or something. So you may want to triple check those files. Also, this pipeline is very sensitive to special characters in names, so if in doubt, keep it simple. When debugging issues, it can help to see exactly which command was actually run. You can see this in the execution report HTML file (find the process using the search box, then column with the command, click to expand), or look at the

.command.sh

file in the work directory of the failed process (usually shown in the error message; you can even use this to manually run the the command).

Thanks for the quick response! I’ll simplify the file names to make sure they don’t contain special characters and see if this solves the problem. I’ll let you know if it works or not.