How to Use HISAT2: Mapping in RNA-Seq Analysis

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 and Bowtie2 . This page explains how to use HISAT2.

Please refer to the RNA-Seq analysis workflow overview.

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).

Check the help message:

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

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

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.

Next, map the read sequences to the reference genome.

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.

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