How to Use Bowtie2: Mapping in RNA-Seq Analysis

Last updated: January 20, 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 mapping software for RNA-Seq includes HISAT2, STAR, and Bowtie2. This page explains how to use Bowtie2.

Please refer to the RNA-Seq analysis workflow overview.

Installation

Bowtie2 can be installed using conda.

$ conda install -c bioconda bowtie2

Let’s display the help message.

$ bowtie2 -h

If you see output like the following, the installation was successful.

bowtie2 -h Bowtie 2 version 2.4.1 by Ben Langmead (langmea@cs.jhu.edu, www.cs.jhu.edu/~langmea) Usage: bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> | --interleaved <i> | -b <bam>} [-S <sam>] <bt2-idx> Index filename prefix (minus trailing .X.bt2). NOTE: Bowtie 1 and Bowtie 2 indexes are not compatible. <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). <i> Files with interleaved paired-end FASTQ/FASTA reads Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2). <bam> Files are unaligned BAM sorted by read name. <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, create an index for the reference sequence using the following command.

$ bowtie2-build -f genome.fa genome

genome.fa is the reference sequence you want to map against, provided as a FASTA file. Compressed files (gzip) are also supported.

This command generates six index files:genome.1.bt2 to genome.4.bt2, 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.

Mapping

Next, map the read sequences to the reference sequence.

$ bowtie2 -x genome -1 reads1.fastq.gz -2 reads2.fastq.gz -S output.sam

This command produces a SAM file. Since SAM files can be large, it is often convenient to convert them to BAM format and sort them as shown below.

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

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

RNA-Seq Data Analysis Software: Accelerate Your Publication

With our RNA-Seq data analysis software, you won't need to outsource or rely on collaborators. You can start analyzing the data yourself right away, without the need for high-spec computers or knowledge of Linux commands.

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.

→ Learn more

Recommended Pages