Bowtie input question - combining left and right reads?

[RNAddict]

Hi all,
I have some 100bp PE reads. I combined my left.fa and right.fa files into one file 'both.fa' and tried aligning these reads to my reference using Tophat/Bowtie.

Alignment ran to completion without reporting any errors, however the accepted_hits.bam output was not usable by cufflinks.

First I got an error saying that the file was not sorted.

After sorting I got an error saying "EOF marker is absent" and that the file is not a BAM file.

Does anyone know:
1. can Bowtie accept both left and right reads in the same file?
2. would this cause sorting/header errors?

[dpryan]

Don't combine the files, bowtie/tophat (most modern aligners, actually) can accept both files simultaneously. See the tophat manual.

By default, tophat sorts the resulting reads by coordinate, so you shouldn't get that error. Given that you ended up getting an EOF error when trying to sort the BAM file yourself, I suspect there was actually an error while tophat was writing its output. You might have luck going through the logs.