Quantification

Quantification with SALMON

The next step in the analysis is to quantify our alignments against a transcriptome with the SALMON software

Soybean alignment script

#!/bin/tcsh

#BSUB -J salmonquant_Portfolio #job name

#BSUB -n 12 #number of threads

#BSUB -W 5:0 #time for job to complete

#BSUB -R "rusage[mem=0.2]" #to request a node with 0.2 GB of memory

#BSUB -o salmonquant_Portfolio.%J.out #output file

#BSUB -e salmonquant_Portfolio.%J.err #error file

#to quantify aligned reads using salmon in quasi indexing mode

#set threads under 12 on Hazel

#working directory path is /share/bitcpt/S23/ajlovele/Portfolio

#input of aligned reads path is /share/bitcpt/S23/ajlovele/Portfolio/AlignedToTranscriptome

#output of aligned reads will go into salmon_align_quant subdirectory in working directory

##########################

# Set the variables

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set SALMON=/usr/local/usrapps/bitcpt/salmon/bin/salmon

set cdna=/share/bitcpt/S23/referenceGenomes/Portfolios/Glycine_max_Wm82-ISU01_v2/glyma.Wm82_ISU01.gnm2_transcriptome.fasta

set IN=/share/bitcpt/S23/ajlovele/Portfolio/AlignedToTranscriptome

##########################

##########################

set s=Gm_OldLeaf_Rep1

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_OldLeaf_Rep2

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_OldLeaf_Rep3

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_OldLeaf_Rep4

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_OldLeaf_Rep5

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_SA_Rep1

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_SA_Rep2

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_SA_Rep3

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_SA_Rep4

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_SA_Rep5 

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_YoungLeaf_Rep1

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_YoungLeaf_Rep2

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_YoungLeaf_Rep3

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_YoungLeaf_Rep4

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

##########################

##########################

set s=Gm_YoungLeaf_Rep5

${SALMON} quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}.quant

The code in the script can be broken down into two general parts. In one part, we define the SALMON, cdna, and IN. variables. These store paths to the salmon executable, our soybean transcriptome file, and the path to the folder that holds our alignments. The second part is the call to the salmon software itself. 

Code components

• quant: Run the salmon software in quantification mode

• -l: Library type

• -a: Path to alignment file

• --targets: Path to transcriptome file

• -o: Path to output directory.

Output

The output from the salmon call includes several files nested within a directory named after each sample. The file that we are most interrested in is the quantification file. This is named quant.sf for each sample. Here's what our output directory looks like:

Rename Soybean quant.sf and export to new directory

Locate all quant.sf files in the salmon_align_quant directory

Then rename and export to quant.sf_export

Example: Rename quant.sf to Gm_OldLeaf_Rep1.quant.sf 

Do this for each replicate and each tissue type.

Move these renamed files using Globus to a new directory called quant.sf_export

These files can be moved to your local computer and then visualized using GALAXY