variant-calling

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Germline and somatic short-variant calling pipeline for Illumina short-read data. Use when the user mentions BWA, BWA-MEM2, GATK, HaplotypeCaller, Mutect2, VCF, GVCF, VQSR, variant calling, germline, somatic, SNV, or indel calling from FASTQ/BAM.

50installs

Sourceawslabs/hcls-agent-skills

Added on2026-10-08

NPX Install

npx skill4agent add awslabs/hcls-agent-skills variant-calling

SKILL.md Content

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Variant Calling (GATK4) — Pipeline Skill (Thin Scaffold)

Overview

Adds decision logic for VQSR vs hard filters, WES vs WGS parameter differences, and GATK4-specific gotchas that LLMs frequently get wrong (filter thresholds, GVCF requirements, reference consistency).

Usage

Activate when choosing between VQSR and hard filters for a given cohort size
Activate when setting up WES vs WGS pipeline parameters
Activate when running Mutect2 tumor/normal or tumor-only somatic calling

Core Concepts

Decision Logic

Germline calling strategy:
├── Single sample, no future joint calling → HaplotypeCaller direct VCF
└── Cohort (≥2 samples) → HaplotypeCaller -ERC GVCF → GenomicsDBImport → GenotypeGVCFs

Filtering strategy:
├── ≥30 WGS samples OR ≥30 WES exomes → VQSR
└── <30 samples OR single sample → Hard filters

Somatic calling:
├── Tumor + matched normal → Mutect2 with both BAMs
└── Tumor-only → Mutect2 + PoN (MANDATORY) + gnomAD germline resource

WES vs WGS parameter differences:

Step	WGS	WES
BQSR / HaplotypeCaller	No `-L`	`-L capture.bed -ip 100`
Known sites for BQSR	Genome-wide	Genome-wide (do NOT subset)
Filtering	VQSR (≥30 samples)	Hard filters unless ≥30 exomes
Joint genotyping intervals	Per-chromosome	`-L capture.bed`

Critical Parameters

Hard filter thresholds (GATK Best Practices):

Filter	SNP threshold	Indel threshold
QD	< 2.0	< 2.0
FS	> 60.0	> 200.0
MQ	< 40.0	—
MQRankSum	< -12.5	—
ReadPosRankSum	< -8.0	< -20.0
SOR	> 3.0	> 10.0

VQSR truth sensitivity levels:

SNPs: 99.7
Indels: 99.0
Indel
```
--max-gaussians 4
```
(fewer training variants than SNPs)

VQSR annotations:

-an QD -an FS -an MQ -an MQRankSum -an ReadPosRankSum -an SOR

Mutect2 essentials:

--germline-resource af-only-gnomad.hg38.vcf.gz

```
--panel-of-normals pon.vcf.gz
```
(essential for tumor-only)
```
--f1r2-tar-gz
```
→
```
LearnReadOrientationModel
```
(FFPE/OxoG artifacts)

GetPileupSummaries

+

CalculateContamination

before

FilterMutectCalls

Common Mistakes

Wrong: Aligning without proper
```
@RG
```
headers (missing ID, SM, PL, LB) Right: Always specify at alignment:
```
-R '@RG\tID:x\tSM:x\tPL:ILLUMINA\tLB:x'
```
Why: GATK refuses to run or silently merges samples when SM tags are wrong
Wrong: Running HaplotypeCaller without
```
-ERC GVCF
```
for cohort analysis Right: Always produce GVCFs when joint genotyping will be performed Why: Regular VCFs cannot be joint-genotyped; must re-call from BAM
Wrong: Reusing SNP hard-filter thresholds for indels (e.g.,
```
FS > 60
```
for indels) Right: Use
```
FS > 200
```
for indels,
```
FS > 60
```
for SNPs Why: Indels tolerate higher strand bias; SNP thresholds over-filter real indels
Wrong: Applying VQSR to <30 samples Right: Use hard filters for small cohorts Why: VQSR needs many variants to train its Gaussian mixture model
Wrong: Subsetting
```
--known-sites
```
VCFs to WES capture BED for BQSR Right: Use genome-wide known-sites; only restrict analysis intervals via
```
-L
```
Why: BQSR needs genome-wide known sites to model base quality errors
Wrong: Skipping
```
samtools index
```
between GATK steps Right: Index after every BAM-producing step Why: GATK requires BAM indices; missing them causes immediate failure
Wrong: Mixing reference files from different genome builds Right: Ensure ref.dict, ref.fa.fai, BWA index, and all VCFs match the same build Why: Mismatched dictionaries cause silent failures or cryptic errors
Wrong: Running Mutect2 tumor-only without a panel of normals Right: Always provide
```
--panel-of-normals
```
for tumor-only calling Why: Without PoN, recurrent sequencing artifacts are called as somatic mutations

Response Format

Lead with the command or code the user needs — explain after
Structure as: confirm inputs → working code → key parameters explained → gotchas
One complete working example per task; do not show every alternative
Keep code comments minimal and functional (what, not why-it-exists)
Target: 50-100 lines of code with brief surrounding explanation