How to Use TopHat2: 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 mapping software for RNA-Seq includes HISAT2, STAR, Bowtie2. This page explains how to use TopHat2.

Note that TopHat2 is an older software tool, so in most cases it is recommended to use HISAT2 or STAR instead.

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

To use TopHat2, a Bowtie2 index is required. If you install TopHat2 using Conda, Bowtie2 will be installed automatically.

Let’s check the Bowtie2 help message.

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

Next, display the TopHat2 help message.

If the following output is shown, TopHat2 is ready to use.

First, create an index of the reference genome using the following command. Here, Bowtie2 is used.

genome.fa is the reference sequence you want to map reads to, provided as a FASTA file. Gzip-compressed files can also be used.

This command generates several files such as genome.1.bt2 through genome.4.bt2, as well as genome.rev.1.bt2 and genome.rev.2.bt2. Index files are required for fast sequence searching and must be created in advance for almost all mapping software, not just Bowtie2.

Next, map the read sequences to the reference genome using TopHat2.

As a result, a BAM file named accepted_hits.bam is generated in theoutput directory.

You can visualize the mapping results using a genome browser such as IGV, as shown below.