Plasmidsaurus RNA-seq Analysis Pipeline

A robust, production-ready bioinformatics pipeline for processing and analyzing RNA-seq data from Plasmidsaurus sequencing services.

Note: This pipeline is for RNA-seq analysis, not plasmid sequencing/verification.

Overview

This pipeline implements a comprehensive RNA-seq workflow from raw FASTQ files through differential expression and functional enrichment analysis. The workflow is designed to be reproducible, well-documented, and suitable for production use.

Pipeline Steps

Step	Tool	Version	Description
1	BCL Convert / fqtk	4.3.6 / 0.3.1	FastQ generation and demultiplexing
2	FastP	0.24.0	Read filtering and QC
3	STAR	2.7.11	Alignment to reference genome
4	samtools	1.22.1	BAM coordinate sorting
5	UMICollapse	1.1.0	UMI-based deduplication
6	RSeQC / Qualimap	5.0.4 / 2.3	Mapping quality control
7	MultiQC	1.32	Comprehensive QC reporting
8	featureCounts	2.1.1	Gene expression quantification
9	edgeR	4.0.16	TMM normalization and sample correlations
10	edgeR	4.0.16	Differential expression analysis
11	GSEApy	0.12	Functional enrichment (MSigDB Hallmark)

Requirements

Software Dependencies

bcl-convert >= 4.3.6
fqtk >= 0.3.1
fastp >= 0.24.0
STAR >= 2.7.11
samtools >= 1.22.1
UMICollapse >= 1.1.0
rseqc >= 5.0.4
qualimap >= 2.3
multiqc >= 1.32
subread >= 2.1.1 (featureCounts)
R >= 4.0
  - edgeR >= 4.0.16
  - DESeq2 (optional)
Python >= 3.8
  - gseapy >= 0.12

Reference Files

• Reference genome (FASTA)
• Gene annotation (GTF)
• STAR genome index

Project Structure

├── scripts/          # Pipeline scripts
├── config/           # Configuration files
├── data/             # Input data (not tracked)
├── results/          # Output results (not tracked)
├── logs/             # Execution logs (not tracked)
├── CLAUDE.md         # Claude Code guidance
└── README.md         # This file

Quick Start

# 1. Clone the repository
git clone <repository-url>
cd mcbla_plasmidsaurus_pipe

# 2. Configure the pipeline
cp config/config.template.yaml config/config.yaml
# Edit config/config.yaml with your paths and parameters

# 3. Run the pipeline
./run_pipeline.sh -i /path/to/fastq -o results/ -c config/config.yaml

Usage

./run_pipeline.sh [OPTIONS]

Options:
  -i, --input DIR       Input directory containing FASTQ files (required)
  -o, --output DIR      Output directory for results (required)
  -c, --config FILE     Configuration file (default: config/config.yaml)
  -t, --threads INT     Number of threads (default: 8)
  -s, --step STEP       Start from specific step (default: 1)
  -h, --help            Display help message

Examples:
  # Run full pipeline
  ./run_pipeline.sh -i data/fastq/ -o results/ -t 16

  # Resume from alignment step
  ./run_pipeline.sh -i data/fastq/ -o results/ -s 3

Output Structure

results/
├── 01_filtered/           # FastP filtered reads
├── 02_aligned/            # STAR alignments
├── 03_sorted/             # Coordinate-sorted BAMs
├── 04_dedup/              # Deduplicated BAMs
├── 05_qc/                 # RSeQC and Qualimap output
├── 06_multiqc/            # MultiQC report
├── 07_counts/             # featureCounts matrices
├── 08_normalization/      # TMM-normalized counts
├── 09_de/                 # Differential expression results
└── 10_enrichment/         # GSEA results

Configuration

See config/config.template.yaml for all available options including:

• Reference genome and annotation paths
• Tool-specific parameters
• Sample metadata
• Contrast definitions for DE analysis

License

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Authors

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Acknowledgments

Pipeline design based on Plasmidsaurus bioinformatics workflows.