Quantification

Login

1. Login into HPC

mcescalo@login.hpc.ncsu.edu

2. Change the working directory to my user name under bitcpt group

>>: cd /share/bitcpt/Fall2022/mcescalo/Portafolio

3. Create "Sl.salmon.sh" shell script using vi command and write the salmon quantification script for Sl

>>>: vi Sl.salmon.sh

#!/bin/tcsh

#BSUB -J salmonquant_Sl_2X #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_Tom.%J.out #output file

#BSUB -e salmonquant_Tom.%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=/gpfs_common/share03/bitcpt/Fall2022/referenceGenomes/Solanum_lycopersicum/Portfolio/Tom-Heinz1706/Tom-Heinz_transcriptome.fasta

set IN=/share/bitcpt/Fall2022/mcescalo/Portafolio/AlignedToTranscriptome

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

# Tom-Leaf 1

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

set s=Sl_Leaf_Rep1_2X

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

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

# Tom-Leaf 3

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

set s=Sl_Leaf_Rep3_2X

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_2X

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_2X

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

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

# TomSAM 3

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

set s=Sl_SAM_Rep3_2X

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

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

# TomSAM 3

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

set s=Sl_SAM_Rep4_2X

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

echo Done

4. Submit the job to run the salmon quantification

>>>: bsub < Sl.salmon.sh

5. After successful completion of the salmon quantification job, change the directory to salmon_align_quant(output directory for star alignment)

>>>: cd salmon_align_quant

6. list out the files inside the starOutputfiles directory using the tree command

>>>: tree

.

├── Sl_Leaf_Rep1_2X.quant

│   ├── aux_info

│   │   ├── ambig_info.tsv

│   │   ├── expected_bias.gz

│   │   ├── fld.gz

│   │   ├── meta_info.json

│   │   ├── observed_bias_3p.gz

│   │   └── observed_bias.gz

│   ├── cmd_info.json

│   ├── libParams

│   │   └── flenDist.txt

│   ├── logs

│   │   └── salmon_quant.log

│   └── quant.sf

├── Sl_Leaf_Rep3_2X.quant

│   ├── aux_info

│   │   ├── ambig_info.tsv

│   │   ├── expected_bias.gz

│   │   ├── fld.gz

│   │   ├── meta_info.json

│   │   ├── observed_bias_3p.gz

│   │   └── observed_bias.gz

│   ├── cmd_info.json

│   ├── libParams

│   │   └── flenDist.txt

│   ├── logs

│   │   └── salmon_quant.log

│   └── quant.sf

├── Sl_SAM_Rep1_2X.quant

│   ├── aux_info

│   │   ├── ambig_info.tsv

│   │   ├── expected_bias.gz

│   │   ├── fld.gz

│   │   ├── meta_info.json

│   │   ├── observed_bias_3p.gz

│   │   └── observed_bias.gz

│   ├── cmd_info.json

│   ├── libParams

│   │   └── flenDist.txt

│   ├── logs

│   │   └── salmon_quant.log

│   └── quant.sf

├── Sl_SAM_Rep2_2X.quant

│   ├── aux_info

│   │   ├── ambig_info.tsv

│   │   ├── expected_bias.gz

│   │   ├── fld.gz

│   │   ├── meta_info.json

│   │   ├── observed_bias_3p.gz

│   │   └── observed_bias.gz

│   ├── cmd_info.json

│   ├── libParams

│   │   └── flenDist.txt

│   ├── logs

│   │   └── salmon_quant.log

│   └── quant.sf

├── Sl_SAM_Rep3_2X.quant

│   ├── aux_info

│   │   ├── ambig_info.tsv

│   │   ├── expected_bias.gz

│   │   ├── fld.gz

│   │   ├── meta_info.json

│   │   ├── observed_bias_3p.gz

│   │   └── observed_bias.gz

│   ├── cmd_info.json

│   ├── libParams

│   │   └── flenDist.txt

│   ├── logs

│   │   └── salmon_quant.log

│   └── quant.sf

└── Sl_SAM_Rep4_2X.quant

├── aux_info

│   ├── ambig_info.tsv

│   ├── expected_bias.gz

│   ├── fld.gz

│   ├── meta_info.json

│   ├── observed_bias_3p.gz

│   └── observed_bias.gz

├── cmd_info.json

├── libParams

│   └── flenDist.txt

├── logs

│   └── salmon_quant.log

└── quant.sf

Interpretation of the Script

salmon quant #To quantify reads using salmon

-l A #library type, -l A allows salmon to automatically infer the library type

-a ${IN}/${s}_Aligned.toTranscriptome.out.bam##path to an input alignment files

-targets ${cdna}#Path to the trancriptomic cDNA fasta file

-o salmon_align_quant/${s}.quant#path to output directory specific to the sample