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