Bowtie2 Tutorial: Read Mapping for RNA-Seq Analysis

Last updated: March 13, 2026

📖 RNA-Seq Data Analysis Workflow — check it out for an overview.

Introduction

Quantifying gene expression from RNA-Seq sequencing data typically requires a mapping step. Mapping is the process of aligning read sequences (FASTQ files) to their corresponding positions on a reference sequence.

Popular mapping tools for RNA-Seq include HISAT2, STAR, and Bowtie2. This page walks through how to use Bowtie2.

For an overview of the entire RNA-Seq data analysis workflow, see the RNA-Seq analysis workflow guide.

Installation

Bowtie2 can be installed via conda:

$ conda install -c bioconda bowtie2

Verify the installation by displaying the help message:

$ bowtie2 -h

If you see output similar to the following, Bowtie2 has been installed successfully.

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)

Before mapping, build an index from the reference sequence with the following command:

$ bowtie2-build -f genome.fa genome

Here, genome.fa is the FASTA file of the reference sequence you want to align reads against. Gzip-compressed files are also accepted.

This produces six index files: genome.1.bt2 through genome.4.bt2, plus genome.rev.1.bt2 and genome.rev.2.bt2. Index files enable fast sequence lookup and must be generated ahead of time for virtually all mapping tools, not just Bowtie2.

Mapping

Now you can align the reads to the reference sequence:

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

This produces a SAM file containing the alignment results. In practice, it is convenient to convert the SAM file to BAM format and sort it for downstream analysis:

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

You can inspect the results in a genome browser such as IGV. The aligned 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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