Hi Jose,


1.       Using xyplot(`FL1-H` ~ `FSC-H` |
> as.factor(Well.Id),transform("FL1-H"=log10) %on% emkFPcg2[1:12],
> displayFilter=TRUE,smooth=FALSE, col=c("red","blue"),
> filter="Negative.Control") I get plots with FL1-H scale from 0 t0 3, but
> all my events are from 2.5 to 3. All the samples have most of the events
> on the first decade.
>
> Why is this happening? I did not have this problem with other
> samples....
>
>
I don't see this behavior in the sample data set, so I'm not sure what's
causing
your problem.  Maybe the events are piling up on the axis?

The plateCore %on% function is a wrapper for the flowCore %on% function.
The function attempts to transform the Negative Control Gates (if they
exist) and
then transform the flowSet/plateSet.  If something were wrong with flowCore
log10 transforms I think we'd see a lot of people reporting problems.


>
> 2.      I do not have negative control in each plate, only in one well
> in one plate, how can I use this for all of the plates? I am running
> batches of 10+ plates, single color, 5K collected. What would the max
> number of plates that could be combined?
>
>
The easiest way might be to give the negative control file a new
name and well id, and then copy the fcs file into each folder.  You'd
have to update the well annotation table to reflect this change.  If
you're using 96-well plates, create an fcs file with the well identifier
of something like I01 so you don't overwrite existing wells.  Alternatively
you could read in the plate and the negative control wells as two
separate flowSets and then combine them before creating a plateSet.
You'd still have to assign the negative control well a new id that
doesn't overlap with anything on the plate.  Lastly, if you know where
you'd like to set your gate, 100 units for example, you could directly
assign the value using

plateSet@wellAnnotation$Negative.Control.Gate <- 100,

which should set the value at 100 for all the wells on the plate.

The maximum number of plates depends upon how many
events are on each plate and how much memory you have
available.  I had no trouble combining 9 plates (1 million
events per plate) on a 64-bit Red Hat machine with
32GB of RAM.  If you filter the plates to remove events
that are not of interest, then you should easily be able
to combine 10-20 plates if you're only collecting 5K per well.


Sincerely,

Errol Strain

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