Salmon-Quant

GOAL!! Quantification of reads

Main Quiz - How many reads come from each gene?

downloaded from

https://salmon.readthedocs.io/en/latest/index.html

Check version 1.4.0

#Description of Salmon

• A module for transcript quantification, it estimates the number of reads in each sample mapping to a reference transcript
• This count is an estimate of the abundance or expression level of these transcripts
• By the way Salmon is an alignment free method, read quantification does not require an input BAM file.
• Salmon uses a selective mapping algorithm to align the reads directly to a set of target transcripts from a reference database

EXPERIMENT !! using Salmon to quantify Anopheles funestus reads (from RNASeq sequencing) that maps to transcripts of AnFun3 reference genome

step 1

download the reference Transcriptome File

download An. funestus genome

wget https://vectorbase.org/common/downloads/Current_Release/AfunestusFUMOZ/fasta/data/VectorBase-57_AfunestusFUMOZ_AnnotatedTranscripts.fasta > /home/nattohz/Fun_RNASeq/Refseq

download An. funestus GFF file

wget http://ftp.ensemblgenomes.org/pub/metazoa/release-53/gff3/anopheles_funestus/Anopheles_funestus.AfunF3.53.gff3.gz > /home/nattohz/Fun_RNASeq/Refseq

Study Design File

Download the study design file from dropbox (in .txt file format)

step 2

Create Transcriptome Index

module load salmon salmon index -t /home/nattohz/Fun_RNASeq/RefseqVectorBase-57_AfunestusFUMOZ_AnnotatedTranscripts.fasta
-i salmon_index -k 31 ls /home/nattohz/Fun_RNASeq/Refseq/salmon_index

Check out the various index files created in the salmon_index folder created by salmon

Step 3

quality control

Trim raw reads to remove illumina adapter sequences, NNNN and bad quality reads

module load trimmomatic trimmomatic Siaya_Res_R1.fastq.gz Siaya_Res_R2.fastq.gz
Siaya_Res_trimmed_forward_paired.fastq.gz Siaya_Res_trimmed_forward_unpaired.fastq.gz
Siaya_Res_trimmed_reverse_paired.fastq.gzSiaya_Res_trimmed_reverse_unpaired.fastq.gz
ILLUMINACLIP:/home/nattohz/adapters/TruSeq3-PE.fa:2:30:10:2:keepBothReads
LEADING:3 TRAILING:3 MINLEN:35

if there are more than two pairs to be trimmed, a simple script will do this as follows

create a script text editor with nano and past the script below, edit the sample names to coincide with appropriate names

nano Trim_raw.sh

#!/usr/bin/env bash for r1 in *_R1.fastq.gz do echo $r1 sample=$(basename $r1) sample=${sample%_R1.fastq.gz} echo "Processing sample: "$sample module load trimmomatic trimmomatic PE ${sample}_R1.fastq.gz ${sample}_R2.fastq.gz
${sample}_trimmed_forward_paired.fq.gz ${sample}_trimmed_forward_unpaired.fq.gz
${sample}_trimmed_reverse_paired.fq.gz ${sample}_trimmed_reverse_unpaired.fq.gz
ILLUMINACLIP:/home/nattohz/adapters/TruSeq3-PE.fa:2:30:10:2:keepBothReads
LEADING:3 TRAILING:3 MINLEN:35

once trimming is done

Confirm QC is acceptabe

with RNASeq data I will worry much about adapters and NNNNN , wont mind so much about overrepresentation

module load fastqc fastqc *_paired.fq.gz module load multiqc multiqc .

check combined records or you can do individually by opening the html file on firefox

Step 4

Transcript Quantificaiton with salmon

use trimmed and paired reads to estimate transcript abundance:

gunzip /home/nattohz/Fun_RNASeq/Refseq/Anopheles_funestus.AfunF3.53.gff3.gz gffread /home/nattohz/Fun_RNASeq/Refseq/Anopheles_funestus.AfunF3.53.gff3 -T -o /home/nattohz/Fun_RNASeq/Refseq/Anopheles_funestus.AfunF3.53.gtf /home/nattohz/Fun_RNASeq/Quantified salmon quant
--geneMap /home/nattohz/Fun_RNASeq/Refseq/Anopheles_funestus.AfunF3.53.gtf
--threads 2 -l A
-i /home/nattohz/Fun_RNASeq/Refseq/salmon_index/VectorBase-57_AfunestusFUMOZ_AnnotatedTranscripts.fasta
-1 ${sample}_trimmed_forward_paired.fq.gz
-2 ${sample}_trimmed_reverse_paired.fq.gz -o /home/nattohz/Fun_RNASeq/Quantified

done

############# DOWNSTREAM ANALYSIS IN R ############### as follows