sh_pipeline_PE.txt

#!/bin/bash

# Adding necessary paths to the environment
export PATH=$PATH:/home/aysegul/Desktop/RNAseqAnalysis/AppFiles/FastQC
export PATH=$PATH:/home/aysegul/Desktop/RNAseqAnalysis/AppFiles/samtools/bin
export PATH=$PATH:/home/aysegul/Desktop/RNAseqAnalysis/AppFiles/hisat2
export PATH=$PATH:/home/aysegul/Desktop/RNAseqAnalysis/AppFiles/subread/bin

# Change working directory to where the analysis will take place
cd /home/aysegul/Desktop/RNAseqAnalysis/

# mkdir -p RNAseqAnalysis/fastqFiles/
# mv -v yourfastq RNAseqAnalysis/fastqFiles/

# STEP 1: FASTQC
echo "FASTQC STARTED!"
fastqc fastqFiles/*.fastq -o fastqFiles  # Check adapter content or quality using fastqc HTML result
echo "FASTQC FINISHED!"

# STEP 2: TRIMMOMATIC
echo "Trimmomatic STARTED!"
read1= "fastqFiles/fastq_R1.fastq"
read2= "fastqFiles/fastq_R2.fastq"
adapters= "AppFiles/Trimmomatic-0.39/adapters"  #Use TruSeq3-PE.fa:2:30:10 adapter for Illumina PE fastq
java -jar /home/aysegul/AppFiles/Trimmomatic-0.39/trimmomatic-0.39.jar PE -phred33 $read1 $read2 \
paired_read1.fastq unpaired_read1.fastq  paired_read2.fastq unpaired_read2.fastq \
ILLUMINACLIP:"$adapters" LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
echo "Trimmomatic FINISHED!"

# STEP 3: HISAT2
echo "Hisat2 STARTED!"
hisat2 -q --rna-strandness FR -x RNAseqAnalysis/HISAT2/grch38/genome -1 paired_read1.fastq -2 paired_read2.fastq \
  | samtools sort -o sorted_fastq_files.bam
echo "Hisat2 FINISHED!"

# STEP 4: featureCounts
echo "featureCounts STARTED!"
featureCounts -p -s 0 -T 4 -a Homo_sapiens.GRCh38.110.gtf -o featureCounts_output.txt sorted_fastq_files.bam
echo "featureCounts FINISHED!"

# Calculate and display elapsed time
duration=$SECONDS
echo "$(($duration / 60)) minutes and $(($duration % 60)) seconds elapsed."