bio-longread-alignment

Version Compatibility

Reference examples tested with: minimap2 2.26+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to 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.

Long-Read Alignment with minimap2

"Align my long reads to the reference" → Map ONT or PacBio reads using minimap2 with technology-specific presets for optimal sensitivity and accuracy.

• CLI: minimap2 -ax map-ont ref.fa reads.fq | samtools sort -o aligned.bam (ONT), minimap2 -ax map-hifi (PacBio HiFi)

Oxford Nanopore Alignment

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

PacBio HiFi Alignment

# 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

# 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

# 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

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)

# 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)

# 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

# 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)

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

Keep Secondary and Supplementary

# 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

# 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

# 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

# Get alignment statistics
samtools flagstat aligned.bam

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

Convert PAF to BED

# 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