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WES/WGS Single-Sample Pipeline

A user-focused guide to processing whole-exome (WES) and whole-genome (WGS) data using GATK Best Practices.

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Diagram: Single-Sample WES/WGS Workflow​

WES/WGS single-sample workflow

Purpose​

This pipeline processes one sample at a time and produces a filtered VCF and gVCF organized for downstream tools and project QC. It automatically adapts to:

  • WES: restricted to an exome interval list
  • WGS: whole genome (no interval restriction)

What the Pipeline Does​

1. Alignment & Read Groups​

  • Align paired-end FASTQ files using BWA-MEM.
  • Add read groups (sample, library, lane, platform) required by GATK.
  • Output: lane-level BAMs with correct RG tags.

2. Lane Merging​

  • Merge all lane BAMs for the same sample into a single BAM.
  • Ensures duplicate marking and BQSR operate on the full dataset.

3. Duplicate Marking​

  • Use GATK MarkDuplicates on the merged BAM.
  • Flags PCR/optical duplicates to prevent them from inflating support for artefactual variants.

4. Base Quality Score Recalibration (BQSR)​

  • Two-step process: BaseRecalibrator then ApplyBQSR.
  • Uses known variant databases (dbSNP, Mills, 1000G indels) to model and correct systematic base-quality errors.
  • Output: recalibrated BAM used for variant calling.

5. Variant Calling (HaplotypeCaller, gVCF)​

  • Run GATK HaplotypeCaller in GVCF mode (-ERC GVCF).
  • WES: uses exome intervals; WGS: full genome.
  • Output: <id>.hc.g.vcf.gz (per-sample gVCF).

6. GenotypeGVCFs (Raw VCF)​

  • Run GATK GenotypeGVCFs on the sample gVCF.
  • Output: <id>.hc.raw.vcf.gz (raw VCF with SNPs and indels).

7. Variant Quality Score Recalibration (VQSR)​

  • If there are enough variants (SNPs and indels), build VQSR models:
    • VariantRecalibrator for SNPs and indels separately.
    • Uses multiple annotations (QD, MQ, FS, MQRankSum, ReadPosRankSum).
  • Output: recalibration VCFs and tranche files.

8. Apply VQSR or Hard Filters​

  • If models exist:
    • Apply SNP VQSR.
    • Then apply INDEL VQSR.
    • Output: <id>.hc.vqsr.vcf.gz.
  • If not:
    • Skip directly to hard filters on the raw VCF or post-SNP VQSR VCF.

9. Generate Final QC VCF​

  • Run VariantFiltration with recommended hard filters on annotations.
  • Output: <id>.hc.QC.vcf.gz (final QC VCF).

10. Coverage & Sex Determination​

  • Extract chromosome 1 reads from raw and recalibrated BAMs.
  • Compute coverage statistics.
  • Infer sample sex from final VCF using a dedicated script.
  • Outputs:
    • 03_stats/<id>.coverage.txt
    • 03_stats/<id>.sex.txt

Output Files​

FileMeaning
02_varcall/<id>.hc.g.vcf.gzPer-sample gVCF (HaplotypeCaller)
02_varcall/<id>.hc.raw.vcf.gzRaw VCF after GenotypeGVCFs
02_varcall/<id>.hc.vqsr.vcf.gzVCF after VQSR (if VQSR was applied)
02_varcall/<id>.hc.QC.vcf.gzFinal QC-filtered VCF (recommended)
03_stats/<id>.coverage.txtCoverage metrics
03_stats/<id>.sex.txtSex determination result
logs/<id>.logMain pipeline log

When to Use This Pipeline​

  • Standard research WES or WGS processing.
  • Generating gVCFs for cohort joint genotyping.
  • Producing filtered single-sample VCFs for downstream review or interpretation.