ViroMap

Targeted virome capture panel design pipeline for hybrid-capture NGS enrichment of vertebrate DNA and RNA viruses from clinical and environmental samples.

---

Overview

ViroMap is a computational probe design pipeline for constructing targeted virome capture panels. The panel targets vertebrate-infecting DNA and RNA viruses with a non-redundant probe set suitable for solution-based hybrid capture workflows. When coupled with standard metagenomic sequencing, this design enables several-hundred-fold enrichment of viral read fractions and markedly improved genome breadth and depth compared to unenriched libraries.

Key applications:

• Comprehensive virome profiling in clinical, surveillance, and research settings
• Sensitive detection of low-abundance or mixed viral infections in high background host material
• Characterization of both known viruses and divergent variants with substantial sequence mismatch to the probe design

---

Repository Structure

ViroMap/
├── cluster_sequences_v2.sh         # CD-HIT based viral sequence clustering (v2)
├── cluster_sequences_v3.sh         # Improved clustering with updated thresholds (v3)
├── design_probes_v3.py             # Probe tiling and design logic from clustered sequences
├── filter_probes_v1.sh             # Post-design probe filtering (GC content, complexity)
├── remove_degenerate_bases.py      # Removes probes containing degenerate IUPAC bases
├── validate_probes_blast_v2.sh     # BLAST-based off-target and specificity validation
└── Degenerate_removal_Summary.md   # Summary report of degenerate base filtering step

---

Pipeline Workflow

Viral Reference Sequences (NCBI/ViPR)
            │
            ▼
  1. Cluster Sequences         (cluster_sequences_v3.sh)
     └─ CD-HIT-EST clustering to reduce redundancy
            │
            ▼
  2. Design Probes             (design_probes_v3.py)
     └─ Sliding-window tiling of representative sequences
            │
            ▼
  3. Filter Probes             (filter_probes_v1.sh)
     └─ GC-content, low-complexity, and length filters
            │
            ▼
  4. Remove Degenerate Bases   (remove_degenerate_bases.py)
     └─ Strict IUPAC base validation
            │
            ▼
  5. Validate Specificity      (validate_probes_blast_v2.sh)
     └─ BLAST against host genome to remove off-target probes
            │
            ▼
       Final Probe Panel (.fasta)

---

Requirements

Tool	Version	Purpose
Python	≥ 3.8	Probe design and degenerate base removal
CD-HIT-EST	≥ 4.8	Sequence clustering
BLAST+	≥ 2.11	Off-target probe validation
Bash	≥ 4.0	Shell scripts

---

Installation

# Clone the repository
git clone https://github.com/rvrane/ViroMap.git
cd ViroMap

# Make shell scripts executable
chmod +x *.sh

# Install Python dependencies
pip install biopython

---

Usage

Step 1: Cluster viral reference sequences

bash cluster_sequences_v3.sh \
  --input viral_references.fasta \
  --output clustered_representatives.fasta \
  --identity 0.90

Step 2: Design probes

python design_probes_v3.py \
  --input clustered_representatives.fasta \
  --probe_length 120 \
  --tiling_offset 60 \
  --output probes_raw.fasta

Step 3: Filter and clean probes

bash filter_probes_v1.sh --input probes_raw.fasta --output probes_filtered.fasta
python remove_degenerate_bases.py --input probes_filtered.fasta --output probes_clean.fasta

Step 4: Validate specificity

bash validate_probes_blast_v2.sh \
  --input probes_clean.fasta \
  --host_db /path/to/hg38_blast_db \
  --output probes_validated.fasta

---

Output

File	Description
clustered_representatives.fasta	Non-redundant viral sequence representatives
probes_raw.fasta	All tiled probes before filtering
probes_filtered.fasta	GC and complexity filtered probes
probes_clean.fasta	Degenerate-base-free probe set
probes_validated.fasta	Final panel after host off-target removal
Degenerate_removal_Summary.md	QC report for degenerate base removal

---

Notes & Limitations

• Probe sensitivity depends on the completeness of the input viral reference database
• CD-HIT clustering identity threshold (--identity) should be tuned based on target viral diversity
• Host off-target validation requires a pre-built BLAST database for the relevant host genome
• Designed for Linux/HPC environments; not tested on Windows natively

---

Future Work

• Integration with Snakemake or Nextflow for end-to-end workflow management
• Docker/Conda containerization
• Automated NCBI viral sequence fetching module
• Support for RNA virus-specific uracil handling

---

Author

Rugved Rane
Senior Bioinformatician | Clinical Genomics
GitHub

---

License

MIT License — see LICENSE for details.