Chapter 6: Step 4 — Variant Calling: Germline vs. Somatic Architecture

Chapter 6: Step 4 — Variant Calling: Germline vs. Somatic Architecture

Variant calling is the computational stage that evaluates recalibrated aligned reads against the human reference genome to identify non-reference mutations. As highlighted in our masterclass architecture, variant calling bifurcates fundamentally into two distinct biological tracks: Germline Workflow and Somatic Oncology Workflow.

The Two Divergent Workflows: Architectural Comparison

Path A: Germline Calling with GATK HaplotypeCaller & DeepVariant

GATK HaplotypeCaller avoids the pitfalls of single-column pileup callers by performing local de novo micro-assembly:

1. Identifies candidate Active Regions showing signs of non-reference variation.
2. Constructs a local de Bruijn graph across the active region to reconstruct candidate haplotypes.
3. Evaluates read alignments against haplotypes using a PairHMM (Hidden Markov Model) to calculate likelihood matrices.
4. Emits a Genomic VCF (GVCF) containing statistical records for both variant and invariant sites.

# Germline Variant Calling in GVCF Mode (GATK HaplotypeCaller)
gatk HaplotypeCaller     -R GRCh38.primary_assembly.fa     -I patient_recalibrated.bam     -O patient_germline.g.vcf.gz     -ERC GVCF

# Genotype GVCF to produce standard multi-sample or single-sample VCF
gatk GenotypeGVCFs     -R GRCh38.primary_assembly.fa     -V patient_germline.g.vcf.gz     -O patient_germline.raw.vcf.gz

Path B: Somatic Oncology Calling with GATK MuTect2

Somatic calling differs fundamentally because tumor samples are mixtures of malignant clones, normal stromal cells, and immune infiltrates. A critical driver mutation (e.g. PIK3CA H1047R) may be present at only 3% Variant Allele Frequency (VAF).

# Somatic Variant Calling with Matched Tumor-Normal Pair (GATK MuTect2)
gatk Mutect2     -R GRCh38.primary_assembly.fa     -I tumor_recalibrated.bam     -I normal_recalibrated.bam     -normal NORMAL_SAMPLE_NAME     --germline-resource af-only-gnomad.hg38.vcf.gz     --panel-of-normals pon_1000g_hg38.vcf.gz     -O somatic_raw.vcf.gz

# Filter Somatic Calls for Cross-Sample Contamination and Orientation Bias
gatk FilterMutectCalls     -R GRCh38.primary_assembly.fa     -V somatic_raw.vcf.gz     -O somatic_filtered.vcf.gz