Bowtie2 is an ultrafast and memory-efficient tool for aligning sequencing reads to long reference sequences. Although Bowtie and Bowtie2 are both fast read aligners, there are few main differences between them:
- Bowtie2 supports gapped alignment with affine gap penalties, without restrictions on the number of gaps and gap lengths.
- Bowtie supports reads longer than 50bp and is generally faster, more sensitive, and uses less memory than Bowtie.
- Bowtie support only end-to-end alignments, while Bowtie2 supports both end-to-end and local alignment.
- Bowtie has an upper limit on read length of around 1,000 bp, while Bowtie2 does not have any.
- Bowtie2’s paired-end alignment is more flexible that Bowtie’s.
- Bowtie2 does not align colorspace reads.
- Bowtie and Bowtie2 indices are not compatible.
Same as Bowtie, the first and basic step of running Bowtie2 is to build Bowtie2 index from a reference genome sequence.The basic usage of thecommandbowtie2-buildis:
$ bowtie2-build -f input_reference.fasta index_prefixwhereinput_reference.fastais an input file of sequence reads in fasta format, andindex_prefixis the prefix of the generated index files. Beside the option -fthat is used when the reference input file is a fasta file, the option -ccan be used when the reference sequences are given on the command line.
The command bowtie2 takes a Bowtie2 index and set of sequencing read files and outputs set of alignments in SAM format. The general bowtie2 usage is:
$ bowtie2 -x index_prefix [-q|--qseq|-f|-r|-c] [-1 input_reads_pair_1.[fasta|fastq] -2 input_reads_pair_2.[fasta|fastq] | -U input_reads.[fasta|fastq]] -S bowtie2_alignments.sam [options]whereindex_prefixis the generated index using thebowtie2-buildcommand, andoptionsare optional parameters that can be found in the Bowtie2 manual. Bowtie2 supports both single-end (input_reads.[fasta|fastq])and paired-end (input_reads_pair_1.[fasta|fastq], input_reads_pair_2.[fasta|fastq]) files in fasta or fastq format. The format of the input files also needs to be specified by using one of the following flags:-q(fastq files), –qseq (Illumina’s qseq format),-f(fasta files), -r (raw one sequence per line), or-c(sequences given on command line).
An example of how to run Bowtie2 local alignment on Swan with paired-end fasta files and 8 CPUs is shown below:
bowtie2_alignment.submit
#!/bin/bash#SBATCH --job-name=Bowtie2#SBATCH --nodes=1#SBATCH --ntasks-per-node=8#SBATCH --time=168:00:00#SBATCH --mem=10gb#SBATCH --output=Bowtie2.%J.out#SBATCH --error=Bowtie2.%J.errmodule load bowtie/2.3bowtie2 -x index_prefix -f -1 input_reads_pair_1.fasta -2 input_reads_pair_2.fasta -S bowtie2_alignments.sam --local -p $SLURM_NTASKS_PER_NODEBowtie2 Output
Bowtie2 outputs alignments in SAM format that can further be manipulated with different tools, like SAMtools and GATK. Each line from the file describes an alignment and is a collection of at least 12 fields separated by tabs. Detailed information about Bowtie2 output fields can be found in the Bowtie2 manual.
