Quantification & Normalization

Script for Quantifying aligned reads using Salmon

#!/bin/tcsh

#BSUB -J salmonquant_At #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_At.%J.out #output file

#BSUB -e salmonquant_At.%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/At

#input of aligned reads path is /share/bitcpt/Fall2022/unityID/At/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/Arabidopsis_thaliana/tair10/Arabidopsis_thaliana.TAIR10.cdna.all.fa.gz

set IN=/share/bitcpt/Fall2022/casjogre/At/AlignedToTranscriptome

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

# At-Leaf 1

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

set s=Col-0_Leaf_Rep1

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

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

# At-Leaf 2

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

set s=Col-0_Leaf_Rep2

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

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

# At SAM 1

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

set s=Col-0_SAM_rep1_L002

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

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

# At SAM 2

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

set s=Col-0_SAM_rep2_L002

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

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

# At SAM 3

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

set s=Col-0_SAM_rep3_L002

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

echo Done

Code Annotation

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

# At-Leaf 1

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

salmon quant

-l A

-a ${IN}/${s}_Aligned.toTranscriptome.out.bam

--targets ${cdna}

-o salmon_align_quant/${s}.quant

1) Call the software (salmon) and then specify which procedure within the software that we want to run (quant).

• quant is the quantification procedure

salmon quant

2) Specify library type

• -l = library flag

• followed by library type. We provided A to tell Salmon to infer the library type automatically (A=automatic)

-l A

3) Specify the alignment file

• -a = alignment file input flag

• followed by the STAR alignment file that we generated

-a ${IN}/${s}_Aligned.toTranscriptome.out.bam

• We set the IN and s variables earlier in the code with

set IN=/share/bitcpt/Fall2022/casjogre/At/AlignedToTranscriptome

set s=Col-0_Leaf_Rep1

• So when we put it all together, it is the same as

• -a /share/bitcpt/Fall2022/casjogre/At/AlignedToTranscriptome/Col-0_Leaf_Rep1_Aligned.toTranscriptome.out.bam

4) Specify the file of the transcriptome targets file

• --targets = transcriptome targets input file flag

• Salmon uses the genome wide transcriptome file instead of a genome assembly for reference and mapping. The instructors have already found and downloaded these files for the class. When working with your organism you will need to find this file and download to your HPC from Ensembl

• We found the transcriptome file for Arabidopsis here: http://ftp.ensemblgenomes.org/pub/plants/release-53/fasta/arabidopsis_thaliana/cdna/

--targets ${cdna}

• We set the cdna variable earlier in the code with:

• set cdna=/share/bitcpt/Fall2022/referenceGenomes/Arabidopsis_thaliana/tair10/Arabidopsis_thaliana.TAIR10.cdna.all.fa.gz

5) Specify what the name the output and where Salmon should save it

• -o = output flag

• path/to/write/the/output/filename.quant

-o salmon_align_quant/${s}.quant

References and Further Reading

1) https://combine-lab.github.io/salmon/getting_started/

2) https://salmon.readthedocs.io/en/latest/salmon.html

2) https://www.nature.com/articles/nmeth.4197

• access through NCSU libraries for free