Bio Igv

IGV Integration

Automated IGV (Integrative Genomics Viewer) snapshot generation for genomic regions with multiple BAM files. Designed for WGS/WES analysis visualization and quality control.

Quick Start

Install

Install IGV:

# macOS
brew install --cask igv

# Linux
wget https://data.broadinstitute.org/igv/projects/downloads/2.16/IGV_Linux_2.16.0_withJava.zip
unzip IGV_Linux_2.16.0_withJava.zip

# Windows
# Download from https://software.broadinstitute.org/software/igv/download

Install Python dependencies:

uv pip install typer

Basic Usage

# Single region with multiple BAM files
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample1.bam sample2.bam sample3.bam \
  --region chr1:1000-2000 \
  --output-dir ./igv_snapshots

# Multiple regions from BED file
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample1.bam sample2.bam \
  --bed regions.bed \
  --output-dir ./igv_snapshots

Scripts

generate_igv_snapshots.py - IGV Snapshot Generation

Generate PNG screenshots of genomic regions with multiple BAM tracks using IGV batch mode.

Required Arguments

• --genome TEXT - Genome assembly ID (e.g., hg38, hg19, mm39)
• --bam PATH - BAM file path(s). Can specify multiple files.
• --region TEXT or --bed PATH - Either single region (e.g., chr1:1000-2000) or BED file with multiple regions
• --output-dir PATH - Output directory for snapshots

Optional Arguments

IGV Configuration:

• --igv-path TEXT - Path to IGV executable (default: auto-detect)
• --max-panel-height INT - Maximum panel height in pixels (default: 500)
• --java-heap TEXT - Java heap size (default: 2g)

Output:

• --save-batch-script - Save IGV batch script for debugging (default: False)

Output Format

PNG Screenshots:

• Named by region: chr1_1000-2000.png
• Or by BED name field: region_name.png

Optional Batch Script (with --save-batch-script):

• igv_batch_script.txt - IGV commands used for snapshot generation

Usage Examples

# Visualize single region with 3 BAM files
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam control.bam treatment1.bam treatment2.bam \
  --region chr1:1000-2000 \
  --output-dir ./snapshots

# Visualize multiple regions from BED file
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam alignment.bam \
  --bed regions_of_interest.bed \
  --output-dir ./snapshots

# Custom IGV settings with larger panel height
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample.bam \
  --region chr1:1000-2000 \
  --output-dir ./snapshots \
  --max-panel-height 800 \
  --java-heap 4g

# Save batch script for debugging
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample.bam \
  --region chr1:1000-2000 \
  --output-dir ./snapshots \
  --save-batch-script

Workflow Examples

Example 1: Visualize BLAT Results

Visualize BLAT alignment results from BAM file:

# Step 1: Extract BLAT hit regions to BED file
# (Assuming you have BLAT results in PSL or JSON format)
echo -e "chr1\t1000\t2000\tinsert1" > blat_hits.bed
echo -e "chr2\t5000\t6000\tinsert2" >> blat_hits.bed

# Step 2: Generate IGV snapshots
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam alignment.bam \
  --bed blat_hits.bed \
  --output-dir ./blat_visualizations

Example 2: Compare Multiple Samples

Visualize the same regions across multiple BAM files:

# Generate snapshots with all samples loaded
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample1.bam sample2.bam sample3.bam \
  --bed candidate_regions.bed \
  --output-dir ./multi_sample_comparison

Example 3: Validate Variant Calls

Visualize variant sites with BAM alignment:

# Step 1: Extract variant positions to BED
# (From VCF file using vcf-toolkit or bcftools)
bcftools query -f '%CHROM\t%POS0\t%END\t%ID\n' variants.vcf > variant_sites.bed

# Step 2: Visualize with flanking regions
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam alignment.bam \
  --bed variant_sites.bed \
  --output-dir ./variant_validation \
  --max-panel-height 600

IGV Batch Script Format

The script generates IGV batch commands in this format:

new
genome hg38
load /path/to/sample1.bam
load /path/to/sample2.bam
snapshotDirectory /path/to/output
maxPanelHeight 500
goto chr1:1000-2000
snapshot chr1_1000-2000.png
goto chr2:5000-6000
snapshot chr2_5000-6000.png
exit

Error Handling

IGV Not Found

$ python scripts/generate_igv_snapshots.py --genome hg38 --bam sample.bam --region chr1:1000-2000 --output-dir ./out

Error: IGV not found. Please install IGV or specify path with --igv-path.
Install: brew install --cask igv (macOS) or download from https://software.broadinstitute.org/software/igv/download

Solution: Install IGV or specify path:

# Install IGV
brew install --cask igv

# Or specify custom path
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample.bam \
  --region chr1:1000-2000 \
  --output-dir ./out \
  --igv-path /path/to/igv.sh

Invalid Region Format

$ python scripts/generate_igv_snapshots.py --genome hg38 --bam sample.bam --region 1000-2000 --output-dir ./out

Error: Invalid region format: 1000-2000. Expected format: chr1:1000-2000

Solution: Use correct region format chr:start-end:

python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample.bam \
  --region chr1:1000-2000 \
  --output-dir ./out

No Snapshots Generated

$ python scripts/generate_igv_snapshots.py --genome hg38 --bam sample.bam --region chr1:1000-2000 --output-dir ./out

Snapshots generated successfully in: ./out
Total regions processed: 1

Warning: No snapshots found. Check IGV output for errors.

Possible causes:

1. BAM file not indexed (create with samtools index)
2. Region not present in BAM file
3. IGV failed to start (check --save-batch-script for debugging)

Solution:

# Check BAM file is indexed
samtools index sample.bam

# Save batch script to debug IGV execution
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample.bam \
  --region chr1:1000-2000 \
  --output-dir ./out \
  --save-batch-script

# Manually run batch script to see errors
igv.sh -b ./out/igv_batch_script.txt

Best Practices

1. Always Index BAM Files

Create BAM index before generating snapshots:

# ✅ Good: Index BAM files first
samtools index sample1.bam
samtools index sample2.bam

# Then generate snapshots
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample1.bam sample2.bam \
  --region chr1:1000-2000 \
  --output-dir ./snapshots

2. Use BED Files for Multiple Regions

For batch processing, use BED files instead of multiple script calls:

# ✅ Good: Single call with BED file
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample.bam \
  --bed regions.bed \
  --output-dir ./snapshots

# ❌ Bad: Multiple script calls
python scripts/generate_igv_snapshots.py --bam sample.bam --region chr1:1000-2000 --output-dir ./out
python scripts/generate_igv_snapshots.py --bam sample.bam --region chr2:5000-6000 --output-dir ./out
# ... (inefficient, IGV starts/stops repeatedly)

3. Adjust Panel Height for Readability

Increase panel height when visualizing multiple BAM tracks:

# ✅ Good: Larger panel for 5 BAM files
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam s1.bam s2.bam s3.bam s4.bam s5.bam \
  --region chr1:1000-2000 \
  --output-dir ./snapshots \
  --max-panel-height 800

4. Use Appropriate Genome Assembly

Match genome assembly to BAM file reference:

# Check BAM header for reference genome
samtools view -H alignment.bam | grep @SQ

# Use matching genome assembly
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam alignment.bam \
  --region chr1:1000-2000 \
  --output-dir ./snapshots

When to Use igv-integration vs Manual IGV

Recommended Workflow:

1. Use igv-integration for batch snapshot generation
2. Use manual IGV for interactive exploration and fine-tuning
3. Combine both for comprehensive analysis

Related Skills

• bam-toolkit - BAM file analysis and read extraction
• vcf-toolkit - VCF variant analysis
• blat-api-searching - BLAT genome mapping to find regions for visualization
• sequence-io - FASTA/FASTQ sequence operations

Troubleshooting

Java Heap Size Errors

If IGV fails with memory errors, increase Java heap size:

python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam large_file.bam \
  --region chr1:1000-2000 \
  --output-dir ./out \
  --java-heap 4g

Chromosome Name Mismatch

Ensure chromosome names match between BAM file and region specification:

# Check chromosome names in BAM
samtools idxstats sample.bam | cut -f1

# Use correct chromosome name
# If BAM uses "1" instead of "chr1":
python scripts/generate_igv_snapshots.py \
  --genome hg38 \
  --bam sample.bam \
  --region 1:1000-2000 \
  --output-dir ./out

BED File Format Errors

Ensure BED file has at least 3 columns (chrom, start, end):

# ✅ Good: Valid BED format
chr1	1000	2000
chr2	5000	6000	region_name

# ❌ Bad: Missing columns
chr1:1000-2000

References

• IGV User Guide
• IGV Batch Commands
• IGV Download