bio-read-alignment-bwa-alignment

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---

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

measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes.

allowed-tools:

• read_file
• run_shell_command

---

BWA-MEM2 Alignment

Build Index

# 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

# 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

# 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

# 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

# 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

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

# 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

# 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)

# 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

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