Dear All, I am not so sure about two Tophat settings. Please help. 1) "Number of mismatches allowed in the initial read mapping" Based on the documantation, my understanding is: the reads are re-aligned to transcriptome/genome if the mismatches in the initial alignment is more than the set number (for example, the default setting is 2). In other words, the re-aligning will continue until the mismatches is equal to or below the set number. Is my understanding correct? If I am right, I have one worry: will Tophat stop re-aligning if the mismatch is below 2 (if I use the default setting). If it is true, the read will not be aligned to where it belongs to (with 0 mismatch). 2) "Number of mismatches allowed in each segment alignment for reads mapped independently" Does this mean that the reads will be cut into segments if the mismatches of alignment is more than the set number? Thanks in advance. Jianguang