GPTomics/bio-read-alignment-bwa-alignment
read-alignment/bwa-alignment/SKILL.md
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.
$ install --global
skillsauthnpx skillsauth add GPTomics/bioSkills bio-read-alignment-bwa-alignmentInstall this skill globally with one command. Works with Claude Code, Cursor, and Windsurf.
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SKILL.md
- 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> --versionthen<tool> --helpto 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
# 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
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.
# 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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