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genome-alignment-qc

Assess alignment quality with coverage depth, mapping rates, insert size, and on-target metrics using samtools, mosdepth, and picard.

personAuthor: TashanworldhubOpenAPI
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Skill: genome-alignment-qc

Use When

  • User wants to evaluate BAM quality before variant calling.
  • User needs to assess coverage depth and uniformity across the genome or target regions.
  • User wants to check mapping rate, duplication rate, and insert size distribution.
  • User needs to calculate on-target rate for whole-exome sequencing (WES).
  • User wants to compare QC metrics across samples to detect batch effects.

Inputs

  • Required:
    • BAM file with index (.bam + .bai).
  • Optional:
    • Target BED file (for WES on-target metrics).
    • Reference FASTA (required for picard metrics).
    • Output directory (default: current directory).
    • Per-base coverage flag (enables mosdepth per-base output; default: off).

Workflow

  1. Run samtools stats and samtools flagstat for mapping summary.
  2. Run samtools idxstats for per-chromosome read counts.
  3. Run mosdepth for coverage distribution (genome-wide, or restricted to target BED).
  4. If target BED provided: compute on-target rate and per-target coverage statistics.
  5. Run picard CollectInsertSizeMetrics for insert size distribution.
  6. Run picard CollectAlignmentSummaryMetrics for detailed alignment statistics.
  7. Compile summary: mapping rate, duplicate rate, mean coverage, coverage uniformity (CV), median insert size, on-target rate (if WES).
  8. Flag metrics outside recommended thresholds (see knowledge/sources/genomics/quality-thresholds.md).

Output Contract

  • samtools stats and samtools flagstat output files.
  • mosdepth coverage files (.mosdepth.global.dist.txt, .per-base.bed.gz if enabled).
  • Picard insert size metrics file and histogram.
  • Picard alignment summary metrics file.
  • Compiled QC summary with key metrics and threshold flags.

Limits

  • samtools, mosdepth, and picard must be installed and available on $PATH.
  • mosdepth per-base output can be very large for WGS (~10 GB for 30x human).
  • Picard requires a Java runtime (Java 8+).
  • Reference FASTA is required for picard metrics but not for samtools/mosdepth.
  • This skill reports metrics for a single BAM; multi-sample comparison is out of scope.
  • Common failure cases:
    • BAM index (.bai) missing or out of date relative to the BAM file.
    • Target BED file using different chromosome naming convention than the BAM (e.g., chr1 vs 1).
    • Picard failing due to missing reference FASTA sequence dictionary (.dict).