RNA-seq Pipeline

This script automates a basic RNA-seq pipeline for processing Illumina reads from sequencing runs, including quality control, alignment, and differential expression analysis. The pipeline assumes that the input files are either paired-end or single-end reads in gzipped FASTQ format, and that the reference genome and annotation file are available in the appropriate directories.

Dependencies

The following tools and software packages are required to run this pipeline:

• FastQC: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
• Trimmomatic: http://www.usadellab.org/cms/?page=trimmomatic
• HISAT2: https://daehwankimlab.github.io/hisat2/download/
• SAMtools: http://www.htslib.org/download/
• featureCounts: http://subread.sourceforge.net/
• DESeq2: https://bioconductor.org/packages/release/bioc/html/DESeq2.html
• R: https://www.r-project.org/
• Python: https://www.python.org/
• GNU Bash: https://www.gnu.org/software/bash/

Usage

1. Set the following variables in the script:

   • FASTQ_DIR: the path to the directory containing the input fastq files
   • GENOME_DIR: the path to the directory containing the reference genome files
   • ANNOTATION_FILE: the path to the GTF annotation file
   • OUTPUT_DIR: the path to the directory where output files will be saved

2. Run the script in a Linux terminal using bash:

   • chmod +x rna_seq_pipeline.sh
   • bash RNAseq_pipeline.sh

Pipeline Steps

• Check that each sample has exactly two input fastq files with the correct naming convention.
• Check whether input fastq files are single-end or paired-end.
• Perform quality control and preprocessing using fastqc and trimmomatic.
• Perform alignment using hisat2.
• Convert the resulting SAM file to BAM format and sort it using samtools.
• Count features using featureCounts.
• Perform differential expression analysis using DESeq2 in R.
• Validate the output using validate.py.

Outputs

The pipeline generates the following output files for each sample:

• fastqc_results/: a directory containing the output from fastqc
• *_aligned_reads.bam: the sorted BAM file containing aligned reads
• *_counts.txt: the feature counts for each gene
• *_validated_results.txt: the validated differential expression results

All output files are saved to the specified OUTPUT_DIR, with the sample name included in the file name.