minimap2

Overview

minimap2 is a versatile sequence alignment tool designed for mapping long noisy reads (Oxford Nanopore, PacBio), short reads, and spliced reads (RNA-seq) to a reference genome. It supports a wide range of presets optimised for different data types (map-ont, map-hifi, map-pb, sr, splice) and is substantially faster than its predecessor minimap while maintaining high accuracy. minimap2 is the de facto standard aligner for long-read sequencing data.

Installation

mamba install -c bioconda minimap2

Basic Usage

Download a reference, index it, align nanopore reads, and produce a sorted BAM file.

# Download and index reference
wget -q https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/005/845/GCA_000005845.2_ASM584v2/GCA_000005845.2_ASM584v2_genomic.fna.gz
gunzip GCA_000005845.2_ASM584v2_genomic.fna.gz
samtools faidx reference.fna

# Align nanopore reads
minimap2 -ax map-ont --MD \
  -R '@RG\tID:sample1\tSM:sample1\tPL:ONT' \
  reference.fna reads.fastq.gz \
  | samtools sort -@ 4 -o aligned.sorted.bam -

samtools index aligned.sorted.bam

Key Parameters

Flag / option	Description
`-a`	Output in SAM format (required when piping to samtools).
`-x`	Preset for the data type. Common values: `map-ont` – Oxford Nanopore reads `map-hifi` – PacBio HiFi reads `map-pb` – PacBio CLR reads `sr` – short (Illumina) reads `splice` – long-read RNA-seq
`--MD`	Include the MD tag for reference base information (needed by some variant callers).
`-R`	Read group header line (e.g., `'@RG\tID:sample1\tSM:sample1\tPL:ONT'`).
`-t`	Number of alignment threads (default 3).
`-d`	Save the index to a file for reuse with large genomes.
`-k`	Minimizer k-mer length (preset-dependent).
`-w`	Minimizer window size (preset-dependent).
`--secondary=no`	Do not output secondary alignments.
`-Y`	Use soft clipping for supplementary alignments.

Expected Output

The pipeline above produces:

aligned.sorted.bam – a coordinate-sorted BAM file with all aligned and unaligned reads.
aligned.sorted.bam.bai – the BAM index for fast random access.

Verify alignment statistics with:

samtools flagstat aligned.sorted.bam

This reports total reads, mapped reads, primary and supplementary alignments, and properly paired reads (if applicable).