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I am using Lexogen's Quantitative Sequencing Pool Analysis tool (here: https://github.com/Lexogen-Tools/quantseqpool_analysis) to analyze R1 and R2 files.

I have been able to successfully run this tool. However, I need to downsample and then normalize these results so I need to do this all again.

To downsample, I put the R1s (the ones generated after idemultiplexing) through the SeqTK_sample tool on UseGalaxy. Afterwards, I ran the QSPA tool with the new R1s and old R2s. I skipped the idemultiplexing part by # out the code. This is generating errors (screenshots are attached). I don't think these errors are from me # out the code but rather from the new R1s with the old R2s. I didn't receive any errors the first time I ran this tool.

I will be normalizing my data with DESeq2 using the summary_unique.tsv file.

If anyone has any tips/suggestions it would be much appreciated. I am sure there is a better way to do this. If you require anything from me just ask!

Cheers,

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    $\begingroup$ I don't know anything about this software in particular, but the traceback in the first screenshot makes it look like a compressed file is truncated. QuantSeqPool calls cutadapt which in a roundabout way uses isal-l which uses isal.igzip which finally gives the "Compressed file ended" error. I'd back up a step and look into what file is being given to cutadapt and why it's truncated. $\endgroup$
    – Jesse
    Nov 19, 2021 at 18:16
  • $\begingroup$ Thanks for the advice, Jesse! I compressed these files and loaded them onto the server. I then unzipped them using jar xf as conventional unzip methods weren't working. Do you think this could be an issue? $\endgroup$
    – Allen Schweickart
    Nov 19, 2021 at 18:51
  • $\begingroup$ Sorry for the delay. I wonder if something went wrong during that process (truncated zip file?) and that might explain why "conventional" tools weren't working for you to unzip. I'd suggest using a checksum program on both your local copy and the server copy (md5sum, sha256sum, whatever's available) and re-uploading any files that don't match. $\endgroup$
    – Jesse
    Nov 26, 2021 at 18:00
  • $\begingroup$ I ended up figuring things out. I will post the solution for others to see. $\endgroup$
    – Allen Schweickart
    Dec 1, 2021 at 15:39

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The issue was that we downsampled with useGalaxy's SeqTK tool prior to running UMI-tools and cutadapt (the 2nd and 3rd process, respectively, of Quantitative Sequencing Pool Analysis).

I tried downsampling with the R1 data, available after cutadapt (located in the "trimmed" folder), and everything ran fine. I believe the issues were caused by the barcode sequences being removed by the SeqTK tool prior to running UMI-tools.

You can (somewhat) customize the QSPA script by #'ing out parts of the script that you don't want to run. In this case, I #'d out Star Aligner, UMI-tools (deduplication), and Feature Counts so that the script would stop after cutadapt. After I downsampled with SeqTK, I ran the script using only the parts that I had previously #'d out.

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