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 
…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
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