In identifying unique reads, if tophat alignment is set to allow mismatches, I assume, that a unique read with single perfect alignment may be tagged as having multiple alignments due to a mismatch acceptance. On the other hand, if tophat alignment is set to disallow any mismatches, even the reads which have single unique alignment with one mismatch will get excluded. Is it possible to set tophat parameters so that only if a read has 0 alignments, then to allow 1 mismatch, if this still yields 0 alignments, then allow 2 mismatches, etc. (until x maximum mismatches to accept is reached)? Or, is this best accomplished after the alignment is made, by filtering the output files (e.g., by alignment quality scores) prior to passing to Cufflinks? Either way, how to accomplish this? thanks.