Skill: RNA-seq Alignment QC

Use When

• User wants to assess RNA-seq alignment quality beyond basic mapping stats
• User needs to check strandedness of a library
• User wants to evaluate gene body coverage (5'-to-3' bias)
• User wants to measure rRNA contamination rate
• User needs to verify read distribution across genomic features (CDS, UTR, intron, intergenic)

Inputs

• Required:
  • BAM file with index (.bam + .bai)
• Optional:
  • BED file of gene models (for RSeQC; available from UCSC or RSeQC downloads)
  • GTF annotation file (for Qualimap)
  • Output directory (default: ./alignment_qc_output)
  • Species/genome build for rRNA intervals

Workflow

1. Run RSeQC infer_experiment.py to determine library strandedness.
2. Run RSeQC geneBody_coverage.py for 5'-to-3' coverage profile across gene bodies.
3. Run RSeQC read_distribution.py for read distribution across genomic features (CDS, UTR, intron, intergenic).
4. Run RSeQC inner_distance.py for insert size distribution (paired-end data).
5. Run Qualimap rnaseq for comprehensive RNA-seq QC metrics.
6. Calculate rRNA rate from Qualimap output or custom rRNA interval overlap.
7. Compile summary: strandedness, gene body coverage shape, rRNA rate, feature distribution, junction saturation.
8. Flag metrics that fall outside recommended thresholds.

Output Contract

• RSeQC strandedness report (text)
• Gene body coverage plot (PDF)
• Read distribution table (text)
• Inner distance plot (PDF, paired-end only)
• Qualimap HTML report
• Compiled QC summary (text)

Limits

• RSeQC and Qualimap must be installed and available on PATH.
• BED gene model file is required for most RSeQC modules (downloadable from UCSC or RSeQC site).
• Qualimap requires Java to be installed.
• Qualimap memory scales with BAM size (approximately 2-4 GB for human genome BAMs).
• Common failure cases: missing BED file, BAM without index, Java not available for Qualimap.