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