Align long reads using minimap2 for Oxford Nanopore and PacBio data. Supports various presets for different read types and applications. Use when aligning ONT or PacBio reads to a reference genome for variant calling, SV detection, or coverage analysis.
Reference examples tested with: minimap2 2.26+, samtools 1.19+
Before using code patterns, verify installed versions match. If versions differ:
<tool> --version then <tool> --help to confirm flagsIf code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
"Align my long reads to the reference" → Map ONT or PacBio reads using minimap2 with technology-specific presets for optimal sensitivity and accuracy.
minimap2 -ax map-ont ref.fa reads.fq | samtools sort -o aligned.bam (ONT), minimap2 -ax map-hifi (PacBio HiFi)# Basic ONT alignment
minimap2 -ax map-ont reference.fa reads.fastq.gz | \
samtools sort -o aligned.bam
samtools index aligned.bam
# PacBio HiFi reads (high accuracy)
minimap2 -ax map-hifi reference.fa reads.fastq.gz | \
samtools sort -o aligned.bam
samtools index aligned.bam
# PacBio CLR (continuous long reads, lower accuracy)
minimap2 -ax map-pb reference.fa reads.fastq.gz | \
samtools sort -o aligned.bam
samtools index aligned.bam
# Build index once
minimap2 -d reference.mmi reference.fa
# Use index for alignment
minimap2 -ax map-ont reference.mmi reads.fastq.gz | samtools sort -o aligned.bam
minimap2 -ax map-ont \
-t 8 \ # Threads
-R '@RG\tID:sample\tSM:sample' \ # Read group
--secondary=no \ # No secondary alignments
--MD \ # Generate MD tag for variants
-Y \ # Use soft clipping for supplementary
reference.fa reads.fastq.gz | \
samtools sort -@ 4 -o aligned.bam
# For direct RNA or cDNA sequencing
minimap2 -ax splice reference.fa reads.fastq.gz | \
samtools sort -o aligned.bam
# Provide known splice junctions
minimap2 -ax splice --junc-bed junctions.bed \
reference.fa reads.fastq.gz | samtools sort -o aligned.bam
# Assembly with ~0.1% divergence
minimap2 -ax asm5 reference.fa assembly.fa > aligned.sam
# Assembly with higher divergence (~5%)
minimap2 -ax asm20 reference.fa assembly.fa > aligned.sam
# PAF format (faster, for quick analysis)
minimap2 -x map-ont reference.fa reads.fastq.gz > alignments.paf
# Keep all alignments (for SV calling)
minimap2 -ax map-ont \
--secondary=yes \
-N 5 \ # Max secondary alignments
reference.fa reads.fastq.gz | samtools sort -o aligned.bam
# During alignment pipeline
minimap2 -ax map-ont reference.fa reads.fastq.gz | \
samtools view -b -q 10 | \ # Min mapping quality 10
samtools sort -o aligned.bam
# Concatenate inputs
minimap2 -ax map-ont reference.fa reads1.fastq.gz reads2.fastq.gz | \
samtools sort -o aligned.bam
# Or use file list
cat file_list.txt | xargs minimap2 -ax map-ont reference.fa | \
samtools sort -o aligned.bam
# Get alignment statistics
samtools flagstat aligned.bam
# Detailed stats
samtools stats aligned.bam | grep ^SN
# Extract alignments to BED
awk 'OFS="\t" {print $6, $8, $9, $1, $12, ($5=="+")?"+":"-"}' alignments.paf > alignments.bed
| Preset | Description | Best For |
|---|---|---|
| map-ont | ONT reads | Nanopore genomic |
| map-hifi | PacBio HiFi | PacBio genomic |
| map-pb | PacBio CLR | PacBio CLR |
| splice | Long RNA reads | cDNA, direct RNA |
| asm5 | Low divergence | Same species assembly |
| asm20 | High divergence | Cross-species assembly |
| sr | Short reads | Illumina (basic) |
| Parameter | Default | Description |
|---|---|---|
| -t | 3 | CPU threads |
| -k | 15 | K-mer size |
| -w | 10 | Minimizer window |
| -a | off | Output SAM |
| -x | none | Preset |
| --secondary | yes | Output secondary |
| -N | 5 | Max secondary alignments |
| --MD | off | Generate MD tag |
| -R | none | Read group header |
| -Y | off | Soft clipping for supplementary |
| Format | Flag | Description |
|---|---|---|
| PAF | (default) | Pairwise Alignment Format |
| SAM | -a | Sequence Alignment Map |
| BAM | -a | samtools | Binary SAM |