Dear all, Cuffdiff determines my fragment lenghts to be 188.34 with Std Dev of 58. I am analyzing a paired-end 100 bases library. Which means that according to tophat the mate-inner-dist should be 188 - (2 * 100) = -12. The sequencing center told me that the fragments' mean length is in fact 320 (which I thought was without the primers), so I initially set mate-inner-dist to 120 and I had 85% of the reads aligned (I got the number of aligned reads with samtools falgstat).

On the other hand, using an mate-inner-dist of -12 and Std. Dev. of 58 produces about 70% aligned reads. I have three issues: 1- If indeed my distance is -12, shouldn't my reads overlap by, on average, 12 bases -- I aligned few thousand sequences and none of them do. 2- I don't understand how come the % of aligned with mate-inner-dist of 120 is larger. 3- Are there any other ways of getting useful statistics about bam alignment other than using samtools (idxstats and flagstat)

Thanks for any suggestions you might be able to provide!

Madi