BWA alignment followed by TopHat

[slny]

Hi,

I'm reading the TopHat manual. Looks like I can only use bowtie if I'd like to run TopHat. Can I run sequence alignments with BWA and then use the BWA output to run TopHat?

Thanks,
Slny

[shurjo]

Simple answer - no. TopHat is linked intrinsically with Bowtie. Running it on BWA output rather than raw sequence data could actually be counterproductive.

[slny]

If so, how could I align the reads from exon junctions if I use BWA? Any other tool(s) should I choose if I use BWA? Bowtie is not good for pair-end sequence alignment.

[shurjo]

One way would be to use BWA to align against (the genome +a set of known splice junctions). Even so, TopHat may still be the logical choice as you can align against the same junctions (option -j) as well as any others that are discovered from your data.

[pbluescript]

slny, BWA followed by Tophat is a nice method for paired RNA reads because BWA works so much better for paired end data. I map with BWA and then get unmapped reads or reads with indels or soft clipping and remap them using Tophat. It works well. I generally get more reads mapping overall, but there is a small decrease in the amount of split reads.

[slny]

Thanks a lot for all the responses. Very helpful.

pbluescript, do you use BWA for sequence alignments, and then re-align the unmapped reads with Bowtie followed with TopHat for exon junctions? But Bowtie is not good for pair-end sequencing, so the identification of exon junctions with TopHat will be influenced.

[pbluescript]

Tophat calls Bowtie, so I don't actually use Bowtie alone for mapping RNA reads. The identification of exon junctions still works well since Tophat finds many split reads.

[slny]

Thanks a lot for your explanation. If I choose bowtie for sequence alignment, should I run everything in TopHat because it also calls Bowtie? If so, I don't need to run a separate Bowtie before I use TopHat.

[pbluescript]

If you use Tophat, you don't need to run Bowtie first. You need to have both installed, but you will only run Tophat.

[slny]

Got it. Thanks a lot!