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bowtie can be used to:

  • index reference FASTA nucleotide genomes/sequences
  • align FASTQ sequencing reads to those genomes/sequences

If you want to align short reads (50bp or less), bowtie is more suitable than bowtie2.

Indexing a reference genome/sequence using bowtie-build

Before aligning reads to a reference genome with bowtie, it must be indexed using bowtie-bui ld. This command will create six files with the extensions .1.ebwt, .2.ebwt, .3.ebwt, .4.ebwt, .rev.1.ebwt, and .rev.2.ebwt. These six files together are the index. Once an index has been created, the original reference genome/sequence is no longer needed to align reads. Here’s an example bowtie2-build command:

$ bowtie-build reference_sequence.fasta index_name

In this command, the reference_sequence.FASTA is the nucleotide FASTA sequence we want to index, and index_name is the name of the index. There will be six files beginning with the index_name in the output directory: index_name.1.ebwt, index_name.2.ebwt, index_name.3.ebwt, index_name.4.ebwt, index_name.rev.1.ebwt, and index_name.rev.2.ebwt. There’s no need to specify any of these files individually in subsequent bowtie commands, the index_name alone is enough to refer to the entire index.

Aligning reads to an indexed genome/sequence using bowtie

Now that the genome has been indexed, FASTQ sequencing reads can be aligned to it. This is done using the bowtie command. Here is an example bowtie2 command:

$ bowtie --no-unal --threads n --sam index_name -1 reads_1.fastq -2 reads_2.fastq output.sam

In this command…

  1. --no-unal is an optional argument, meaning reads that do not align to the reference genome will not be written to sam output
  2. --threads is the number (n) of processors/threads used
  3. --sam specifies that the output should be written in the SAM format
  4. index_name is the name of the genome index
  5. -1 is the file(s) containing mate 1 reads (reads_1.fastq)
  6. -2 is the file(s) containing mate 2 reads (reads_2.fastq)
  7. output.sam is the output alignment in sam format


In this video, bowtie-build is used to index S_cere_GCF_000146045.2_R64_genomic.fna, which is a copy of the Saccharomyces cerevisiae S288C genome from RefSeq. The bowtie command is then used to align Saccharomyces cerevisiae RNAseq reads to this bowtie index.


Further reading

  1. The bowtie manual: