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!