How to Use HISAT2: Mapping in RNA-Seq Analysis

Last updated: February 2, 2026

Introduction

When quantifying gene expression levels using sequencing data obtained from RNA-Seq analysis, a mapping step is generally required. Mapping refers to the process of aligning read sequences (FASTQ files) to matching positions on a reference sequence. Commonly used software for RNA-Seq mapping includes HISAT2, STAR, Bowtie2. This page explains how to use HISAT2.

If you would like to understand the overall workflow of RNA-Seq analysis, please refer to the RNA-Seq analysis workflow overview.

Installation

Precompiled binaries are available here, so download the appropriate one for your system.

Unzip the file and add it to your PATH (example below is for macOS).

$ unzip hisat2-2.2.1-OSX_x86_64.zip

Check the help message:

$ hisat2 -h

If the following output is displayed, the installation was successful.

HISAT2 version 2.2.1 by Daehwan Kim (infphilo@gmail.com, www.ccb.jhu.edu/people/infphilo) Usage: hisat2 [options]* -x <ht2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <sam>] <ht2-idx> Index filename prefix (minus trailing .X.ht2). <m1> Files with #1 mates, paired with files in <m2>. Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2). <m2> Files with #2 mates, paired with files in <m1>. Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2). <r> Files with unpaired reads. Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2). <SRA accession number> Comma-separated list of SRA accession numbers, e.g. --sra-acc SRR353653,SRR353654. <sam> File for SAM output (default: stdout) <m1>, <m2>, <r> can be comma-separated lists (no whitespace) and can be specified many times. E.g. '-U file1.fq,file2.fq -U file3.fq'. ...

Index construction (build)

First, build an index for the reference sequence using the following command:

$ hisat2-build genome.fa genome

genome.fa is the FASTA file of the reference sequence you want to map against.

This command generates eight files: genome.1.ht2 through genome.8.ht2. Index files are required for fast string searching and must be created in advance for almost all mapping software, not just HISAT2.

Mapping

Next, map the read sequences to the reference genome.

$ hisat2 -x genome -1 reads1.fastq.gz -2 reads2.fastq.gz -S output.sam

This command outputs a SAM file, which contains the mapping results. It is often convenient to convert the SAM file to a BAM file and sort it, as shown below.

$ samtools view -bS output.sam > output.bam $ samtools sort output.bam > output.sorted.bam

You can visualize the results using a genome browser such as IGV, where the mapped reads will appear as shown below.

RNA-Seq Data Analysis Software

This is an RNA-Seq Data Analysis Software recommended for those who:

✔︎ Seeking to avoid outsourcing or collaboration for RNA-Seq data analysis.

✔︎ Lacking time to learn RNA-Seq data analysis.

✔︎ Frustrated by the complexity of existing tools.

Users can perform gene expression quantification, identification of differentially expressed genes, gene ontology(GO) analysis, pathway analysis, as well as drawing volcano plots, MA plots, and heatmaps.

About the Author

BxINFO LLC

A research support company specializing in bioinformatics.

We provide tools and information to support life science research, with a focus on RNA-Seq analysis.

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