Hi Mark,
From reading the createBeadSummaryData help page and the vignettes, I
gather that you recommend using BASH to detect outliers, and then
using
"mean" in log2-space to summarize the data.
If I plan to use vst/quantile to normalize my data, what would be the
best option for processing the bead level data? I like the idea of
summarizing in log2-space but I want a non-log-space result to use
with
vst. Is there a simple way to accomplish this?
And while I'm asking this, I seem to remember hearing that you get
better results without using the backgroundCorrect step. Is this
recommended?
Many thanks,
Cei
Hi Cei,
You can summarize on the unlogged scale by using the option log=FALSE
in createBeadSummaryData. We're still looking into the best
combination of outlier removal to use with BASH, it could be that the
Illumina method for removing outliers by a 3 MAD cut-off is still
best. This is the default option in beadarray (option
method="Illumina").
One final thing, If you plan to use VST method you'll need to
calculate detection scores prior to running VST, this can be done by:-
Detection(BSData) = calculateDetection(BSData)
The type of background correction we don't recommend is the background
subtraction performed by BeadStudio that subtracts the negative
controls from each bead-type average on an array. This causes a huge
increase in variance and missing values if you try and do a
log-transformation. However, using the combination of VST and quantile
as you plan should take care of these problems.
Hope this helps,
Mark
On Tue, May 19, 2009 at 2:25 PM, Cei Abreu-Goodger <cei at="" ebi.ac.uk="">
wrote:
> Hi Mark,
>
> From reading the createBeadSummaryData help page and the vignettes,
I gather
> that you recommend using BASH to detect outliers, and then using
"mean" in
> log2-space to summarize the data.
>
> If I plan to use vst/quantile to normalize my data, what would be
the best
> option for processing the bead level data? I like the idea of
summarizing in
> log2-space but I want a non-log-space result to use with vst. Is
there a
> simple way to accomplish this?
>
> And while I'm asking this, I seem to remember hearing that you get
better
> results without using the backgroundCorrect step. Is this
recommended?
>
> Many thanks,
>
> Cei
>
Hi Mark,
Thanks for your suggestions. I think I will be using the
method="illumina" for now then.
I've also decided to do vst + rsn, for the simple reason that after an
vst/rsn normalization I can use the inverseVST function in the lumi
package to get the real "un-logged" expression numbers back in case
they're needed (for fold-change calculation, for example).
Thanks again,
Cei
Mark Dunning wrote:
> Hi Cei,
>
> You can summarize on the unlogged scale by using the option
log=FALSE
> in createBeadSummaryData. We're still looking into the best
> combination of outlier removal to use with BASH, it could be that
the
> Illumina method for removing outliers by a 3 MAD cut-off is still
> best. This is the default option in beadarray (option
> method="Illumina").
>
> One final thing, If you plan to use VST method you'll need to
> calculate detection scores prior to running VST, this can be done
by:-
>
> Detection(BSData) = calculateDetection(BSData)
>
> The type of background correction we don't recommend is the
background
> subtraction performed by BeadStudio that subtracts the negative
> controls from each bead-type average on an array. This causes a huge
> increase in variance and missing values if you try and do a
> log-transformation. However, using the combination of VST and
quantile
> as you plan should take care of these problems.
>
> Hope this helps,
>
> Mark
>
> On Tue, May 19, 2009 at 2:25 PM, Cei Abreu-Goodger <cei at="" ebi.ac.uk=""> wrote:
>> Hi Mark,
>>
>> From reading the createBeadSummaryData help page and the vignettes,
I gather
>> that you recommend using BASH to detect outliers, and then using
"mean" in
>> log2-space to summarize the data.
>>
>> If I plan to use vst/quantile to normalize my data, what would be
the best
>> option for processing the bead level data? I like the idea of
summarizing in
>> log2-space but I want a non-log-space result to use with vst. Is
there a
>> simple way to accomplish this?
>>
>> And while I'm asking this, I seem to remember hearing that you get
better
>> results without using the backgroundCorrect step. Is this
recommended?
>>
>> Many thanks,
>>
>> Cei
>>
