[BioC] What should happen to control probe information in beadlevel Illumina analyses?

Johnstone, Alice Alice.Johnstone at esr.cri.nz
Mon Mar 10 02:56:36 CET 2008

Thanks for your reply Simon,
Yes the expression level is at the low end of the range so I do not
think that this is a hybridisation problem as the original control plots
looked fine.
I used the VST transformation with a quantile normalisation of the Bead
Summary data. 
My question is whether it is correct to remove the control probe
information from the Bead Summary object before fitting the regression
model so I don't get any spurious results like I have found, and what is
the best way to do this?

The expression values for one of the control genes are below, the
trt1-control contrast comes up in my topTable results.  This only occurs
when I do my own bead-level summation, the BeadStudio produced bead
summary data does not have the intensity info for this probe.
Trt 1		Control	Trt 2
7.544901	7.638846	7.531172
7.515447	7.614039	7.581452
7.495771	7.605357	7.555899
7.485725	7.622735	7.588895
7.538404	7.592215	7.516454


-----Original Message-----
From: bioconductor-bounces at stat.math.ethz.ch
[mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of Simon Lin
Sent: Saturday, 8 March 2008 5:11 a.m.
To: bioconductor at stat.math.ethz.ch
Subject: [BioC] What should happen to control probe information in
beadlevel Illumina analyses?

Hi Alice,

Please check the expression level of these negative control probes. They
must be very low. If all the negative control probes are high, you might
get a hybridizatin problem; check the stringency control probes.

If these low-expressed negative controls are in your Top50 lists, you
might try a VST transformation method as reported in

Good luck!

Simon Lin
Date: Fri, 7 Mar 2008 16:48:02 +1300
From: "Johnstone, Alice" <Alice.Johnstone at esr.cri.nz>
Subject: [BioC] What should happen to control probe information in bead
level Illumina analyses?
To: "bioconductor" <bioconductor at stat.math.ethz.ch>
<92B672A208AB2F419334EE1EC8C58D9D01D13400 at kscmail1.esr.cri.nz>
Content-Type: text/plain; charset="us-ascii"

Hi again!

When running through my analysis of bead summary data that I have
created from bead level data (background corrected with normexp, and
summarised standard) I produced a topTable list which contains probeIDs
that are not found in Illumina's reference file.  When I checked the
controlprobeprofile.txt there they were!
It looks like I haven't got huge DE change in my experiment, but I still
wouldn't have expected Illumina's negative controls to make the top 50..
Somethings not right.

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