Final Student Portfolio

Overview

In this step, I will be using SALMON to run the process of figuring out the differences between my samples and the reference genome. This process is known as quantification. This process allows me to prepare my data in a way that it can be statistically analyzed and visualized.

What is Salmon?

Salmon is a tool for quantifying the expression of transcripts using RNA-seq data. Salmon uses new algorithms (specifically, coupling the concept of quasi-mapping with a two-phase inference procedure) to provide accurate expression estimates very quickly (i.e. wicked-fast) and while using little memory. Salmon performs its inference using an expressive and realistic model of RNA-seq data that takes into account experimental attributes and biases commonly observed in real RNA-seq data.

References:

Patro, R., Duggal, G., Love, M. I., Irizarry, R. A., & Kingsford, C. (2017). Salmon provides fast and bias-aware quantification of transcript expression. Nature Methods.

Salmon Script for Quantifying aligned reads using Salmon for Tomato

pwd

/share/bitcpt/Fall2022/mmohamm8/Portfolio

## copy script to the current directory

cp /share/bitcpt/Fall2022/mmohamm8/Tom/Tom.salmon.sh Cher.salmon.sh

vi CHer.salmon.sh

#!/bin/tcsh

#BSUB -J salmonquant_Cher #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_Cher.%J.out #output file

#BSUB -e salmonquant_Cher.%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/Portfolio

#input of aligned reads path is /share/bitcpt/Fall2022/UnityID/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/Tom-Cherry

set IN=/share/bitcpt/Fall2022/mmohamm8/Portfolio/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

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

# 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

##Look at the quant files

ll salmon_align_quant

or

ls -lh salmon_align_quant