Quick start: align paired-end FASTQs

This page walks through the two-command workflow: index the reference once, then align reads.

Switching from bwa or bwa-mem2? The command line is unchanged, but rebuild the index if you’re coming from bwa v1 (a bwa-mem2 index is reused as-is), and expect output that is functionally equivalent but not byte-identical. See Coming from bwa or bwa-mem2.

Index the reference

bwa-mem3 index ref.fa

This produces four index files alongside ref.fa:

(No ref.fa.0123 is written: mem reconstructs reference bases from .pac on demand. Pass index --emit-unpacked-ref if a tool such as bwa-mem2 needs it.)

Indexing hg38 takes a few minutes on a multi-core host (construction is multi-threaded and memory-bounded — see Indexing the reference); the final index is roughly 11 GB on disk. The index is read once per mem invocation; for workloads that align many samples, load it into shared memory first (see Quick start: shared-memory index).

Align paired-end reads

bwa-mem3 mem -t 16 ref.fa R1.fq.gz R2.fq.gz > out.sam

-t 16 sets the thread count to 16. bwa-mem3 scales well up to the number of physical CPU cores; hyperthreading provides diminishing returns above that point. See User Guide — Threading and resource use for recommendations at different core counts.

The default output is SAM on stdout. To write BAM directly, add --bam (uncompressed by default — best for piping to samtools sort; use --bam=6 for BGZF-compressed BAM):

bwa-mem3 mem --bam -t 16 ref.fa R1.fq.gz R2.fq.gz \
  | samtools sort -@ 8 -o out.bam -
samtools index out.bam

Tip — Prefer BAM output in production

Piping BAM (--bam) to samtools sort avoids the text formatting and parsing overhead of SAM on both sides of the pipe. For large cohorts this yields a measurable wall-clock reduction. See Best Practices — Output format for the recommended pipeline and a discussion of when SAM is still useful.

Read group tagging

For downstream tools that require a @RG header (most variant callers), pass -R:

bwa-mem3 mem -t 16 \
  -R '@RG\tID:sample1\tSM:sample1\tPL:ILLUMINA\tLB:lib1' \
  ref.fa R1.fq.gz R2.fq.gz > out.sam

The value is a tab-delimited string following BWA conventions. Every aligned record receives an RG:Z: tag matching the ID field of the read-group header.

bwa-mem3 emits the standard SAM tags plus the fork’s HN:i tag; for the full list see Output: SAM/BAM, headers, tags (and Methylation SAM tags under --meth).

See also: Quick start: methylation alignment · Quick start: shared-memory index · User Guide — Aligning short reads · CLI Reference — mem