Hi Tania,
The data is RNA? Did you filter for properly mapped pairs first ('Filter
SAM')? I am guessing not, and that is where the error about single end
data is coming from, and possibly other insert size values that are
skewing the mean.
If you plan on using the Tuxedo pipeline, you might be able to skip
Picard and just use sample of the data to obtain this number. The tool
authors suggest just running Tophat on a sample (few hundred pairs), as
explained in the link below. A mean (and other calculations) can be
generated on any tabular column of data using the tools 'Group',
'Compute', or 'Summary Statistics' (use the tool search to find these in
the tool panel):
http://tophat.cbcb.umd.edu/faq.shtml#mate_inner_dist
Hopefully one of these options works for you,
Jen
Galaxy team
On 2/10/14 8:03 AM, Dottorini, Tania wrote:
Hi,
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
Tania
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Jennifer Hillman-Jackson
http://galaxyproject.org
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