Stringtie invalid escape sequence

I am pretty new to RNA-seq analysis and Galaxy so bare with me.
I have a set of RNA-seq files that I have first put through Trimmomatic and then through HISAT2 for alignment. Everything is well until then and I used the appropriate output options in HISAT2 to make the bam compatible with Stringtie.
Im running Stringtie and asking it to output Gene Counts, Transcript Counts, Assembled transcripts and Gene abundance estimates. I also provided a GTF file for mouse (mm10).
However, for every sample, I get the same standard error:
/usr/local/bin/ SyntaxWarning: invalid escape sequence ‘-’
RE_COVERAGE=re.compile(‘cov “([-+\d.]+)”’)
/usr/local/bin/ SyntaxWarning: invalid escape sequence ‘-’
RE_GFILE=re.compile(‘-G\s*(\S+)’) #assume filepath without spaces…

Whats more, for some of the samples, Gene abundance estimates and assembled transcripts still turn out ok but Gene and Transcript counts return this error. For other samples, none of the 4 files succeed all returning the same error.
Anyone has an idea of what I did wrong?

Welcome, @platonm

Does this prior answer address the problem? Issue with StringTie Output File when using DESeq2 option - #5 by jennaj

And, you might need to double check your reference data. It seems that either the GTF isn’t a match for the reference genome, or, more likely, there is some format problem (headers?). This guide has more details about what to check when using these tools → FAQ: Extended Help for Differential Expression Analysis Tools

Please give those a review and let us know if you solve this. For more troubleshooting here, we’ll need to see what you have so far. The banner at this forum has instructions, or see here directly:

Let’s start here! :slight_smile:


Another person reported this with an example so I was able to take a closer look and confirm where the problem is likely happening for anyone who encounters this same error for the use case.

SyntaxWarning: invalid escape sequence

When that results from running HISAT2 inputs through Stringtie, you’ll need to rerun the mapping again using the advanced option right under where the reference annotation is input. Choose the option to report for Stringtie so the tool can interpret your data.


Screen Shot 2024-05-15 at 6.23.15 PM

This tutorial has an example protocol with exact instructions. You may want to consider the other suggested HISAT2 parameters for scientific reasons. → Hands-on: De novo transcriptome reconstruction with RNA-Seq / Transcriptomics (mapping step)

Hope this helps! :rocket: