Bowtie 2

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Frequently Asked Questions

How is Bowtie 2 different from Bowtie 1?

Bowtie 1 was released in 2009 and was geared toward aligning the relatively short type of sequencing reads (up to 50 bp) prevalent at the time. Since then, sequencing technology has improved both in terms of throughput (more bp produced per sequencer per day) and in terms of read length (more bp per read). Chief differences between Bowtie 1 and Bowtie 2 are:

• Bowtie 2 fully supports gapped alignment with affine gap penalties. Number of gaps and gap lengths are not restricted, except via the user-supplied scoring scheme. Bowtie 1 only finds ungapped alignments.
• For reads longer than about 50 bp Bowtie 2 is generally faster, more sensitive, and uses less memory than Bowtie 1. For relatively short reads (e.g. less than 50 bp) Bowtie 1 is sometimes faster and/or more sensitive.
• Bowtie 2 supports a "local" alignment mode, which doesn't require that reads align end-to-end. This produces alignments that might be "trimmed" (or "soft clipped") at one or both extremes in a way that optimizes alignment score. Bowtie 2 also supports an "end-to-end" alignment mode which, like Bowtie 1, requires that the read align entirely.
• There is no upper limit on read length in Bowtie 2. Bowtie 1 had an upper limit of around 1000 bp.
• Bowtie 2 does away with Bowtie 1's notion of alignment "stratum". In Bowtie 2 all alignments lie along a continuous spectrum of alignment scores.
• There is no longer a distinction between "end-to-end" and "Maq-like" modes as in Bowtie 1. There is just one scoring scheme, similar to Needleman-Wunsch and Smith-Waterman.
• Bowtie 2's paired-end alignment mode is more flexible than Bowtie 1's. For example, for pairs that do not align in a paired fashion, it will attempt to find unpaired alignments for each mate.
• Bowtie 2 does not align colorspace reads.

Is Bowtie 2 a straightforward upgrade from Bowtie 1?

Bowtie 2 is not a "drop-in" replacement for Bowtie 1. Bowtie 2's command-line arguments and genome index format are similar to, but different from Bowtie 1's, so that simply running the Bowtie 2 executable instead of the Bowtie 1 executable will not work.

Are Bowtie 2 and Bowtie 1 genome indexes compatible?

No. Bowtie 2 indexes are formatted differently. Bowtie 1 indexes do not work with Bowtie 2. Bowtie 2 indexes do work with Bowtie 1 as of version 1.2.3.

Does Bowtie 2 supersede Bowtie 1?

Mostly, but not entirely. If your reads are shorter than 50 bp, you might want to try both Bowtie 1 and Bowtie 2 and see which gives better results in terms of speed and sensitivity. In our experiments, Bowtie 2 is generally superior to Bowtie 1 for reads longer than 50 bp. For reads shorter than 50 bp, Bowtie 1 may or may not be preferable.

I used Bowtie 2 to align my paired-end data and very few of my paired-end reads align concordantly. What could have happened?

This is usually because Bowtie 2's paired-end options have been been set to properly reflect how the sequencing library was prepared. See the Paired-end Options section of the manual.

How many mismatches and gaps does Bowtie 2 allow?

Bowtie 2 using a scoring scheme that assigns various scores to matches, mismatches, gap opens, and gap extensions. These are all user-configurable. An alignment with a total score that rises above a user-configurable threshold are considered "valid" and eligible for reporting. For information on how to adjust these settings, see the Scoring Options section of the manual.