Quantification and Normalization

Objective

Salmon software was used to quantified aligned Heinz 1706 sequences into counts to be analyzed downstream.

Heinz Quantification

Copying Salmon script to working directory:

Code:

cp /share/bitcpt/Fall2022/scripts/At.salmon.sh /share/bitcpt/Fall2022/UnityID/Heinz/Heinz.salmon.sh

Edit Heinz Script:

vi Heinz.Salmon.sh

Submit Tomato Quantification Job:

>>>: bsub < Tom.salmon.sh

Salmon Script

#!/bin/tcsh

#BSUB -J salmonquant_Tom #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_Heinz.%J.out #output file

#BSUB -e salmonquant_Heinz.%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/unityID/Heinz

#input of aligned reads path is /share/bitcpt/Fall2022/unityID/Heinz/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/Tom-Heinz1706/Tom-Heinz_transcriptome.fasta

set IN=/share/bitcpt/Fall2022/UnityID/Heinz/AlignedToTranscriptome

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

# 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

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

# 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

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

# Tom 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

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

# Tom 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

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

# Tom SAM 3

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

set Sl_SAM_Rep3_3X

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

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

# Tom 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