Hi All, I'm new to RNA-seq analysis and someone recommended that I do the quantification of my transcripts via Salmon before DGE analysis. I got the Salmon documentation and got the quant.sf files for samples using command line and then input that data into iDep tool for DE analysis. I also knew about Galaxy so I carried out the Salmon quant pipeline in Galaxy and fed the output into DESeq2. I realised that the DGE lists for the top 10 up/down-regulated genes were different in DESeq2 and iDep. I then fed the Salmon quant data from Galaxy into iDep into found different results to the previous analysis where the Salmon quant data had been obtained from command line. I know DESeq2 uses TPM data and iDep uses Read count data but the main thing I noticed was that the TPM and NumReads column values were not the same between the command line generated files and the Galaxy ones. I used the same transcriptome file for both Salmon quantifications. The bash for the command line was:

#!/bin/bash

for fn in *R1_001.fastq.gz

do

base=`basename $fn`;

samp=${base%_R1_001.fastq.gz};

echo "Processing sample ${samp}"

salmon quant -i Ovis_aries_index -l A -1 ${samp}_R1_001.fastq.gz -2 ${samp}_R2_001.fastq.gz 

-p 8 --validateMappings -o Salmon_output/quants/${samp}

done

Why am I getting different outputs between Salmon command line and Salmon on Galaxy?