Fastq To Variants

---
name: bio-workflows-fastq-to-variants
description: End-to-end DNA sequencing workflow from FASTQ files to variant calls. Covers QC, alignment with BWA, BAM processing, and variant calling with bcftools or GATK HaplotypeCaller. Use when calling variants from raw sequencing reads.
tool_type: cli
primary_tool: bcftools
workflow: true
depends_on:
  - read-qc/fastp-workflow
  - read-alignment/bwa-alignment
  - alignment-files/alignment-sorting
  - alignment-files/duplicate-handling
  - variant-calling/variant-calling
  - variant-calling/filtering-best-practices
qc_checkpoints:
  - after_qc: "Q30 >85%, adapter content <1%"
  - after_alignment: "Mapping rate >95%, properly paired >90%"
  - after_dedup: "Duplication rate <30% for WGS, <50% for exome"
  - after_calling: "Ti/Tv ratio ~2.1 for WGS, dbSNP overlap >95%"
---

## Version Compatibility

Reference examples tested with: BWA-MEM2 2.2.1+, Ensembl VEP 111+, GATK 4.5+, bcftools 1.19+, fastp 0.23+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:
- CLI: `<tool> --version` then `<tool> --help` to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.

# FASTQ to Variants Workflow

**"Call variants from my whole-genome or exome FASTQ files"** -> Orchestrate fastp QC, BWA-MEM2 alignment, duplicate marking, BQSR, GATK HaplotypeCaller variant calling, and VQSR/hard filtering to produce filtered VCF output.

Complete pipeline from raw DNA sequencing FASTQ files to filtered variant calls.

## Workflow Overview

```
FASTQ files
    |
    v
[1. QC & Trimming] -----> fastp
    |
    v
[2. Alignment] ---------> bwa-mem2
    |
    v
[3. BAM Processing] ----> sort, markdup, index
    |
    v
[4. Variant Calling] ---> bcftools (primary) or GATK
    |
    v
[5. Filtering] ---------> Quality filters
    |
    v
Filtered VCF
```

## Primary Path: BWA + bcftools

### Step 1: Quality Control with fastp

```bash
# Single sample
fastp -i sample_R1.fastq.gz -I sample_R2.fastq.gz \
    -o sample_R1.trimmed.fq.gz -O sample_R2.trimmed.fq.gz \
    --detect_adapter_for_pe \
    --qualified_quality_phred 20 \
    --length_required 50 \
    --html sample_fastp.html

# Batch processing
for sample in sample1 sample2 sample3; do
    fastp -i ${sample}_R1.fastq.gz -I ${sample}_R2.fastq.gz \
        -o trimmed/${sample}_R1.fq.gz -O trimmed/${sample}_R2.fq.gz \
        --detect_adapter_for_pe \
        --html qc/${sample}_fastp.html
done
```

**QC Checkpoint 1:** Check fastp reports
- Q30 bases >85% (DNA typically higher quality than RNA)
- Adapter content <1%
- No unusual GC distribution

### Step 2: BWA-MEM2 Alignment

```bash
# Index reference (once)
bwa-mem2 index reference.fa

# Align with read group info
for sample in sample1 sample2 sample3; do
    bwa-mem2 mem -t 8 \
        -R "@RG\tID:${sample}\tSM:${sample}\tPL:ILLUMINA\tLB:lib1" \
        reference.fa \
        trimmed/${sample}_R1.fq.gz \
        trimmed/${sample}_R2.fq.gz \
    | samtools view -bS - > aligned/${sample}.bam
done
```

**QC Checkpoint 2:** Check alignment stats
```bash
samtools flagstat aligned/${sample}.bam
```
- Mapped reads >95%
- Properly paired >90%

### Step 3: BAM Processing

```bash
for sample in sample1 sample2 sample3; do
    # Sort by coordinate
    samtools sort -@ 8 -o aligned/${sample}.sorted.bam aligned/${sample}.bam

    # Mark duplicates (samtools method)
    samtools fixmate -m aligned/${sample}.sorted.bam - | \
        samtools sort -@ 8 - | \
        samtools markdup -@ 8 - aligned/${sample}.markdup.bam

    # Index
    samtools index aligned/${sample}.markdup.bam

    # Cleanup intermediate
    rm aligned/${sample}.bam aligned/${sample}.sorted.bam
done
```

**QC Checkpoint 3:** Check duplication rate
```bash
samtools flagstat aligned/${sample}.markdup.bam | grep "duplicates"
```
- WGS: <30% duplicates
- Exome/targeted: <50% duplicates

### Step 4: Variant Calling with bcftools

```bash
# Single sample calling
bcftools mpileup -Ou -f reference.fa aligned/sample1.markdup.bam | \
    bcftools call -mv -Oz -o variants/sample1.vcf.gz

# Multi-sample calling (joint calling)
bcftools mpileup -Ou -f reference.fa \
    aligned/sample1.markdup.bam \
    aligned/sample2.markdup.bam \
    aligned/sample3.markdup.bam | \
    bcftools call -mv -Oz -o variants/cohort.vcf.gz

bcftools index variants/cohort.vcf.gz
```

### Step 5: Variant Filtering

```bash
# Basic quality filter
bcftools filter -Oz \
    -e 'QUAL<20 || DP<10 || MQ<30' \
    -o variants/cohort.filtered.vcf.gz \
    variants/cohort.vcf.gz

# More stringent filter
bcftools filter -Oz \
    -e 'QUAL<30 || DP<10 || DP>200 || MQ<40 || MQB<0.1' \
    -s "LowQual" \
    -o variants/cohort.filtered.vcf.gz \
    variants/cohort.vcf.gz

# Stats
bcftools stats variants/cohort.filtered.vcf.gz > variants/vcf_stats.txt
```

**QC Checkpoint 4:** Check variant stats
- Ti/Tv ratio ~2.1 for whole genome
- Ti/Tv ratio ~2.8-3.0 for exome
- >95% overlap with dbSNP for known sites

## Alternative Path: BWA + GATK HaplotypeCaller

### Step 4 Alternative: GATK Variant Calling (DRAGEN Mode -- Recommended)

```bash
gatk CreateSequenceDictionary -R reference.fa
samtools faidx reference.fa

# DRAGEN-GATK mode: no BQSR needed, improved STR handling and QUAL calibration
gatk HaplotypeCaller \
    -R reference.fa \
    -I aligned/sample1.markdup.bam \
    -O variants/sample1.g.vcf.gz \
    -ERC GVCF \
    --dragen-mode
```

### Step 4 Alternative: GATK Standard Mode (with BQSR)

```bash
gatk BaseRecalibrator \
    -R reference.fa \
    -I aligned/sample1.markdup.bam \
    --known-sites dbsnp.vcf.gz \
    -O recal_data.table

gatk ApplyBQSR \
    -R reference.fa \
    -I aligned/sample1.markdup.bam \
    --bqsr-recal-file recal_data.table \
    -O aligned/sample1.recal.bam

gatk HaplotypeCaller \
    -R reference.fa \
    -I aligned/sample1.recal.bam \
    -O variants/sample1.g.vcf.gz \
    -ERC GVCF

# Joint genotyping (for multiple samples)
gatk GenomicsDBImport \
    -V variants/sample1.g.vcf.gz \
    -V variants/sample2.g.vcf.gz \
    -V variants/sample3.g.vcf.gz \
    --genomicsdb-workspace-path genomicsdb \
    -L intervals.bed

gatk GenotypeGVCFs \
    -R reference.fa \
    -V gendb://genomicsdb \
    -O variants/cohort.vcf.gz
```

### Step 5 Alternative: GATK Variant Filtering

```bash
# Hard filtering (for small cohorts)
gatk VariantFiltration \
    -R reference.fa \
    -V variants/cohort.vcf.gz \
    --filter-expression "QD < 2.0" --filter-name "LowQD" \
    --filter-expression "FS > 60.0" --filter-name "HighFS" \
    --filter-expression "MQ < 40.0" --filter-name "LowMQ" \
    --filter-expression "MQRankSum < -12.5" --filter-name "LowMQRS" \
    --filter-expression "ReadPosRankSum < -8.0" --filter-name "LowRPRS" \
    -O variants/cohort.filtered.vcf.gz

# VQSR (for large cohorts >30 samples)
gatk VariantRecalibrator \
    -R reference.fa \
    -V variants/cohort.vcf.gz \
    --resource:hapmap,known=false,training=true,truth=true,prior=15.0 hapmap.vcf.gz \
    --resource:omni,known=false,training=true,truth=false,prior=12.0 omni.vcf.gz \
    --resource:1000G,known=false,training=true,truth=false,prior=10.0 1000G.vcf.gz \
    --resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp.vcf.gz \
    -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR \
    -mode SNP \
    -O cohort.snp.recal \
    --tranches-file cohort.snp.tranches

gatk ApplyVQSR \
    -R reference.fa \
    -V variants/cohort.vcf.gz \
    -O variants/cohort.vqsr.vcf.gz \
    --recal-file cohort.snp.recal \
    --tranches-file cohort.snp.tranches \
    -mode SNP \
    --truth-sensitivity-filter-level 99.5
```

## Parameter Recommendations

| Step | Parameter | WGS | Exome/Targeted |
|------|-----------|-----|----------------|
| bwa-mem2 | -t | 8-16 | 8 |
| samtools markdup | - | Required | Required |
| bcftools mpileup | -d | 250 (default) | 1000 |
| bcftools mpileup | -q | 20 | 20 |
| bcftools filter | QUAL | >20 | >30 |
| bcftools filter | DP | >10, <2x mean | >20 |
| GATK | intervals | - | Target BED |

## Choosing a Variant Caller

| Criterion | bcftools | GATK HaplotypeCaller | DeepVariant |
|-----------|----------|---------------------|-------------|
| Speed | Fastest | Moderate | Slowest (without GPU) |
| SNP accuracy | Good | Good | Highest |
| Indel accuracy | Moderate (weak in homopolymers) | Good (local assembly) | Highest |
| Joint calling | Basic multi-sample | GVCF workflow (scales to 100k+) | GLnexus (scales to 250k+) |
| Best for | Quick analysis, non-model organisms | Clinical pipelines, GATK ecosystem | Highest accuracy, GPU available |
| VQSR | Not supported | For large cohorts (>30 samples) | Not needed (use GLnexus) |
| DRAGEN mode | N/A | Recommended for single-sample (skips BQSR) | N/A |

For human germline: DeepVariant provides the highest accuracy. GATK DRAGEN-mode is the recommended standard-mode alternative (no BQSR needed). bcftools is best for exploratory work, non-model organisms, or when computational resources are limited.

## Troubleshooting

| Issue | Likely Cause | Solution |
|-------|--------------|----------|
| Low mapping rate | Wrong reference, contamination | Verify reference genome version |
| High duplication | PCR over-amplification, low input | Check library prep, may need more input DNA |
| Low Ti/Tv | False positives | Increase quality filters |
| Missing variants | Too stringent filters, low depth | Relax filters, check coverage |
| Many indels at homopolymers | Sequencing errors | Filter homopolymer regions |

## Complete Pipeline Script

```bash
#!/bin/bash
set -e

# Configuration
THREADS=8
REF="reference.fa"
SAMPLES="sample1 sample2 sample3"
OUTDIR="results"

mkdir -p ${OUTDIR}/{trimmed,aligned,variants,qc}

echo "=== Step 1: QC with fastp ==="
for sample in $SAMPLES; do
    fastp -i ${sample}_R1.fastq.gz -I ${sample}_R2.fastq.gz \
        -o ${OUTDIR}/trimmed/${sample}_R1.fq.gz \
        -O ${OUTDIR}/trimmed/${sample}_R2.fq.gz \
        --detect_adapter_for_pe \
        --html ${OUTDIR}/qc/${sample}_fastp.html \
        -w ${THREADS}
done

echo "=== Step 2: Alignment with bwa-mem2 ==="
for sample in $SAMPLES; do
    bwa-mem2 mem -t ${THREADS} \
        -R "@RG\tID:${sample}\tSM:${sample}\tPL:ILLUMINA" \
        ${REF} \
        ${OUTDIR}/trimmed/${sample}_R1.fq.gz \
        ${OUTDIR}/trimmed/${sample}_R2.fq.gz | \
    samtools view -@ ${THREADS} -bS - > ${OUTDIR}/aligned/${sample}.bam
done

echo "=== Step 3: BAM Processing ==="
for sample in $SAMPLES; do
    samtools fixmate -@ ${THREADS} -m ${OUTDIR}/aligned/${sample}.bam - | \
    samtools sort -@ ${THREADS} - | \
    samtools markdup -@ ${THREADS} - ${OUTDIR}/aligned/${sample}.markdup.bam
    samtools index ${OUTDIR}/aligned/${sample}.markdup.bam
    rm ${OUTDIR}/aligned/${sample}.bam
done

echo "=== Step 4: Joint Variant Calling ==="
bcftools mpileup -Ou -f ${REF} ${OUTDIR}/aligned/*.markdup.bam | \
    bcftools call -mv -Oz -o ${OUTDIR}/variants/cohort.vcf.gz
bcftools index ${OUTDIR}/variants/cohort.vcf.gz

echo "=== Step 5: Filtering ==="
bcftools filter -Oz \
    -e 'QUAL<20 || DP<10 || MQ<30' \
    -o ${OUTDIR}/variants/cohort.filtered.vcf.gz \
    ${OUTDIR}/variants/cohort.vcf.gz
bcftools index ${OUTDIR}/variants/cohort.filtered.vcf.gz

echo "=== Stats ==="
bcftools stats ${OUTDIR}/variants/cohort.filtered.vcf.gz > ${OUTDIR}/variants/stats.txt

echo "=== Pipeline Complete ==="
echo "Filtered VCF: ${OUTDIR}/variants/cohort.filtered.vcf.gz"
```

## Related Skills

- database-access/sra-data - Pull public FASTQ for reanalysis (ENA mirror or STRIDES cloud)
- database-access/ncbi-datasets-cli - Pull reference genome assembly via Datasets v2 CLI
- database-access/ensembl-rest - Pull annotated reference and Compara orthologs for cross-species variants
- read-qc/fastp-workflow - Detailed QC options
- read-alignment/bwa-alignment - BWA-MEM2 parameters
- alignment-files/duplicate-handling - Duplicate marking details
- variant-calling/variant-calling - bcftools calling options
- variant-calling/gatk-variant-calling - GATK HaplotypeCaller details
- variant-calling/filtering-best-practices - Advanced filtering strategies
- variant-calling/variant-annotation - Annotate variants with VEP