Long-Read Alignment with minimap2

Oxford Nanopore Alignment

bash

# Basic ONT alignment
minimap2 -ax map-ont reference.fa reads.fastq.gz | \
    samtools sort -o aligned.bam
samtools index aligned.bam

PacBio HiFi Alignment

bash

# 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 Alignment

bash

# 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

Pre-Build Index for Multiple Runs

bash

# 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

Common Options

bash

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

Splice-Aware Alignment (RNA)

bash

# For direct RNA or cDNA sequencing
minimap2 -ax splice reference.fa reads.fastq.gz | \
    samtools sort -o aligned.bam

With Junction BED (Known Splice Sites)

bash

# Provide known splice junctions
minimap2 -ax splice --junc-bed junctions.bed \
    reference.fa reads.fastq.gz | samtools sort -o aligned.bam

Assembly to Reference Alignment

bash

# 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

Output PAF (Faster, No BAM)

bash

# PAF format (faster, for quick analysis)
minimap2 -x map-ont reference.fa reads.fastq.gz > alignments.paf

Keep Secondary and Supplementary

bash

# 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

Filter Alignments

bash

# 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

Multiple FASTQ Files

bash

# 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

Output Statistics

bash

# Get alignment statistics
samtools flagstat aligned.bam

# Detailed stats
samtools stats aligned.bam | grep ^SN

Convert PAF to BED

bash

# Extract alignments to BED
awk 'OFS="\t" {print $6, $8, $9, $1, $12, ($5=="+")?"+":"-"}' alignments.paf > alignments.bed

Key Presets

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)

Key Parameters

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

Output Formats

Format	Flag	Description
PAF	(default)	Pairwise Alignment Format
SAM	-a	Sequence Alignment Map
BAM	-a \| samtools	Binary SAM

Related Skills

•medaka-polishing - Polish consensus with medaka
•structural-variants - Call SVs from alignments
•alignment-files/sam-bam-basics - BAM manipulation