Salmon normalization and quantification tool
Transcripts per million or TPM metric is used for normalization eliminating the bias of less depth of sequences and shorter length of sequences. Salmon is the quantification tool that produces the counts for each sequence referred to as the read counts.
Quantification of the aligned sequences producing the quant.sf file for each sample which is converted to txt files and used for differential expression analysis
Script below:
#!/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=20000]" #to request a node with 20MB 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 Henry2
#working directory path is /share/bitcpt/Fall2022/maharry/Portfolio
#input of aligned reads path is /share/bitcpt/Fall2022/maharry/Portfolio/AlignedToTranscriptome
#output of aligned reads will go into salmon_align_quant subdirectory in working directory
module load conda
conda activate /usr/local/usrapps/bitcpt/salmon
##########################
# Set the variables
##########################
set cdna=/share/bitcpt/Fall2022/referenceGenomes/Solanum_lycopersicum/Portfolio/Pot-Landrace/Diploid-potato_
transcriptome.fasta
set IN=/share/bitcpt/Fall2022/maharry/Portfolio/AlignedToTranscriptome (path to input files)
##########################
# Sl-Leaf 1
##########################
set s=Sl_Leaf_Rep1_3X
salmon quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}
.quant (path to call software Salmon and specify library flag to be inferred automatically and -a indicating alignment file input)
##########################
# Sl-Leaf 2
##########################
set s=Sl_Leaf_Rep2_3X
salmon quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}
.quant
##########################
# Sl-Leaf 3
############################
set s=Sl_Leaf_Rep3_3X
salmon quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}
.quant
##########################
# Sl-SAM 1
##########################
set s=Sl_SAM_Rep1_3X
salmon quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}
.quant
##########################
# Sl-SAM 2
##########################
set s=Sl_SAM_Rep2_3X
salmon quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}
.quant
##########################
# Sl-SAM 3
##########################
set s=Sl_SAM_Rep3_3X
salmon quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}
.quant
##########################
# Sl-SAM 4
############################
set s=Sl_SAM_Rep4_3X
salmon quant -l A -a ${IN}/${s}_Aligned.toTranscriptome.out.bam --targets ${cdna} -o salmon_align_quant/${s}
.quant
echo Done