Bwa Alignment

---
name: bio-read-alignment-bwa-alignment
description: Align DNA short reads to reference genomes using bwa-mem2, the faster successor to BWA-MEM. Use when aligning DNA short reads to a reference genome.
tool_type: cli
primary_tool: bwa-mem2
---

## Version Compatibility

Reference examples tested with: GATK 4.5+, 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.

# BWA-MEM2 Alignment

**"Align reads with BWA"** → Map DNA reads to a reference genome using BWA-MEM2, the standard aligner for whole-genome and exome sequencing.
- CLI: `bwa-mem2 mem -t 8 ref.fa R1.fq R2.fq | samtools sort -o aligned.bam`

## Build Index

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

# Creates: reference.fa.0123, reference.fa.amb, reference.fa.ann, reference.fa.bwt.2bit.64, reference.fa.pac
```

## Basic Alignment

```bash
# Paired-end reads
bwa-mem2 mem -t 8 reference.fa reads_1.fq.gz reads_2.fq.gz > aligned.sam

# Single-end reads
bwa-mem2 mem -t 8 reference.fa reads.fq.gz > aligned.sam
```

## Alignment with Read Groups

```bash
# Add read group information (required for GATK)
bwa-mem2 mem -t 8 \
    -R '@RG\tID:sample1\tSM:sample1\tPL:ILLUMINA\tLB:lib1' \
    reference.fa reads_1.fq.gz reads_2.fq.gz > aligned.sam
```

## Direct to Sorted BAM

```bash
# Pipe to samtools for sorted BAM output
bwa-mem2 mem -t 8 \
    -R '@RG\tID:sample1\tSM:sample1\tPL:ILLUMINA' \
    reference.fa reads_1.fq.gz reads_2.fq.gz | \
    samtools sort -@ 4 -o aligned.sorted.bam -

# Index the BAM
samtools index aligned.sorted.bam
```

## Mark Duplicates Pipeline

**Goal:** Produce a duplicate-marked, sorted BAM file from raw reads in a single streaming pipeline.

**Approach:** Pipe BWA-MEM2 output through samtools fixmate (to add mate score tags), coordinate sort, and markdup in a single command chain to avoid intermediate files.

```bash
# Full pipeline: align, fixmate, sort, markdup
bwa-mem2 mem -t 8 -R '@RG\tID:sample1\tSM:sample1\tPL:ILLUMINA' \
    reference.fa reads_1.fq.gz reads_2.fq.gz | \
    samtools fixmate -m -@ 4 - - | \
    samtools sort -@ 4 - | \
    samtools markdup -@ 4 - aligned.markdup.bam

samtools index aligned.markdup.bam
```

## Common Options

```bash
bwa-mem2 mem -t 8 \         # Threads
    -M \                     # Mark shorter split hits as secondary (Picard compatible)
    -Y \                     # Use soft clipping for supplementary alignments
    -K 100000000 \           # Process INT input bases in each batch
    -R '@RG\tID:s1\tSM:s1' \ # Read group
    reference.fa r1.fq r2.fq
```

## Key Parameters

| Parameter | Default | Description |
|-----------|---------|-------------|
| -t | 1 | Number of threads |
| -k | 19 | Minimum seed length |
| -w | 100 | Band width for extension |
| -r | 1.5 | Re-seeding trigger ratio |
| -c | 500 | Skip seeds with more than INT hits |
| -A | 1 | Match score |
| -B | 4 | Mismatch penalty |
| -O | 6 | Gap open penalty |
| -E | 1 | Gap extension penalty |
| -M | off | Mark secondary alignments |

## Output Filters

```bash
# Filter unmapped and low quality
bwa-mem2 mem -t 8 reference.fa r1.fq r2.fq | \
    samtools view -@ 4 -bS -q 20 -F 4 - | \
    samtools sort -@ 4 -o aligned.filtered.bam -
```

## Split Read Alignment

```bash
# For SV detection, use -Y for soft clipping
bwa-mem2 mem -t 8 -Y reference.fa r1.fq r2.fq > aligned.sam
```

## Memory Requirements

- Index loading: ~10GB for human genome
- Per thread: ~1-2GB
- Typical human WGS: 30-50GB RAM with 8 threads

## BWA-MEM (Alternative)

```bash
# Build index
bwa index reference.fa

# Paired-end alignment
bwa mem -t 8 reference.fa reads_1.fq.gz reads_2.fq.gz > aligned.sam

# With read groups
bwa mem -t 8 -R '@RG\tID:sample1\tSM:sample1\tPL:ILLUMINA' \
    reference.fa reads_1.fq.gz reads_2.fq.gz > aligned.sam

# Direct to sorted BAM
bwa mem -t 8 -R '@RG\tID:sample1\tSM:sample1\tPL:ILLUMINA' \
    reference.fa reads_1.fq.gz reads_2.fq.gz | \
    samtools sort -@ 4 -o aligned.sorted.bam -
```

## BWA-MEM vs BWA-MEM2

| Feature | BWA-MEM | BWA-MEM2 |
|---------|---------|----------|
| Status | Active | Archived |
| Speed | 1x | 2-3x faster |
| Index format | .bwt | .bwt.2bit.64 |
| Results | Baseline | Nearly identical |
| Memory | ~5GB | ~10GB |

## Related Skills

- read-qc/fastp-workflow - Preprocess reads before alignment
- alignment-files/alignment-sorting - Post-alignment processing
- alignment-files/duplicate-handling - Mark duplicates
- variant-calling/variant-calling - Call variants from BAM