I am trying to determine the mean inner distance between mate pairs, but encountered odd results. Briefly, to calculate the mean inner distance I mapped PE data with 2x100bp read length with Bowtie against the reference transcriptome, and used the Picard InsertSize Metrics to calculate the Mean insert size. I then, subtracted the combined insert size (2*100) from the Mean insert size value, thus obtaining the mean inner distance between mate pairs. In all the cases studied, 4 samples and 4 controls, I have always obtained negative mean inner distance between mate pairs values. In addition, in some cases I had the following error running the Picard Tool:

"Unable to find expected pdf file /galaxy/main/jobdir/006/471/.…../InsertSizeHist.pdf

…This always happens if single ended data was provided to this tool"

But in all cases I can confirm I provided paired-end NGS data. For same of the runs that gave me this failure log, I rerun them again but mapping to the genome instead of the transcriptome, and in this case it did work but always giving me negative distance values.

I would like to know if this is the correct procedure to be followed or if there are other approaches I can use to find these distance values and if I can eventually use such negative distance values in Tophat 


Thank you for your help