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Quick Start Guide

Get your RNA-seq analysis pipeline running in minutes! This guide provides the essential steps to start using the pipeline.

🚀 Prerequisites Check

Before starting, ensure you have:

  • Linux-based operating system
  • Docker installed and running
  • Nextflow 22.04+ installed
  • At least 48 CPU cores and 192GB RAM available
  • Input FASTQ files ready
  • Reference files prepared

⚡ Quick Installation

# 1. Clone the repository
git clone https://github.com/uclanelsonlab/nl-rna-seq_wf.git
cd nl-rna-seq_wf/

# 2. Verify Nextflow installation
nextflow -version

# 3. Verify Docker
docker --version

🔧 Quick Configuration

  1. Update Docker images in nextflow.config:
params {
    bwa_docker = "your-registry/bwa:latest"
    star_docker = "your-registry/star:latest"
    fastp_docker = "your-registry/fastp:latest"
    // ... update other images
}
  1. Prepare reference files:
    • rRNA reference genome
    • Globin RNA reference
    • Human reference genome (FASTA + index)
    • Gencode GTF annotation
    • STAR indices for different read lengths
    • Kallisto transcript index

🏃‍♂️ Quick Run

Minimal Command

nextflow run main.nf \
    --fastq_r1 /path/to/sample_R1.fastq.gz \
    --fastq_r2 /path/to/sample_R2.fastq.gz \
    --sample_name SAMPLE001 \
    --rrna_reference /path/to/rrna.fa \
    --globinrna_reference /path/to/globin.fa \
    --gencode_gtf_path /path/to/gencode.v43.gtf \
    --human_fasta /path/to/GRCh38.fa \
    --star_index_151 /path/to/star_151 \
    --kallisto_index /path/to/kallisto.idx

Test Run

# Run with test data (if available)
nextflow run main.nf -profile test

# Or run with minimal parameters to check setup
nextflow run main.nf --help

📊 Expected Outputs

After successful completion, you'll find:

results/
├── QC/                    # Quality control reports
├── ALIGNMENT/            # STAR alignment files
├── COUNTS/               # Gene expression counts
├── QUANT/                # Kallisto quantification
├── IR/                   # IRFinder results
├── MOSDEPTH/             # Coverage analysis
└── BAM2SJ/              # Splice junction analysis

🐛 Quick Troubleshooting

Common Issues & Solutions

  1. "Docker daemon not running"

    sudo systemctl start docker
sudo usermod -aG docker $USER
newgrp docker

  2. "Out of memory"

    • Increase memory in nextflow.config
    • Use -resume to restart from last successful step

  3. "Reference file not found"

    • Check file paths and permissions
    • Verify file formats and integrity

  4. "Process failed"

    # Check logs
nextflow log

# Resume from last successful step
nextflow run main.nf -resume [session_id]

Debug Mode

# Run with detailed logging
nextflow run main.nf -debug

# Check pipeline status
nextflow log

📈 Performance Tips

  • STAR alignment: Use 48+ cores and 192GB+ RAM
  • Parallel processing: Run multiple samples simultaneously
  • Storage: Ensure sufficient disk space for intermediate files
  • Monitoring: Use htop or top to monitor resource usage

🔄 Resume & Restart

# List available sessions
nextflow log

# Resume specific session
nextflow run main.nf -resume [session_id]

# Clean up and restart
rm -rf work/ results/
nextflow run main.nf [parameters]

📞 Need Help?

✅ Success Checklist

  • Pipeline runs without errors
  • All output directories created
  • Quality control reports generated
  • Alignment files produced
  • Quantification results available
  • Analysis outputs complete

🎯 You're all set! The pipeline should now be processing your RNA-seq data. Monitor the progress and check outputs as they become available.