Hello,
I'm quite new to Bioconductor/affy, and I was wondering if there's a
simple
way to include the absent/present call for a gene in the outputfile
generated
with write.exprs(eset, file='boo') in theaffy package.
the eset was generated with
eset <- expresso(cel, bgcorrect.method = 'rma', normalize.method =
'qspline', pmcorrect.method = 'pmonly',
summary.method='liwong')
For further analyses I'd like to exclude genes that are absent in all
chips.
thanks a lot for your help,
Arne
<arne.muller@aventis.com> writes:
> Hello,
>
> I'm quite new to Bioconductor/affy, and I was wondering if there's a
simple
> way to include the absent/present call for a gene in the outputfile
generated
> with write.exprs(eset, file='boo') in theaffy package.
>
> the eset was generated with
>
> eset <- expresso(cel, bgcorrect.method = 'rma', normalize.method =
> 'qspline', pmcorrect.method = 'pmonly',
> summary.method='liwong')
>
> For further analyses I'd like to exclude genes that are absent in
all chips.
That's tough. It isn't clear what a sensible definition of absent
is. Or present.
Do you mean "expressed" ? "Differentially expressed" ? "sort of
differentially expressed but not too weakly expressed?". For any of
these, you'll need a precise definition (there isn't any in
Bioconductor), and you can compute your own.
(I know that MAS will make these calls; I'm only familiar with Rosetta
Resolver's variant, and they don't really make sense to me -- to be
precise, I know numerically how they are derived, but fail to why they
realistically connect biologically or technologically without a great
deal of assumptions and a wild imagination).
best,
-tony
--
rossini@u.washington.edu
http://www.analytics.washington.edu/
Biomedical and Health Informatics University of Washington
Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer Research
Center
UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is unreliable
FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
CONFIDENTIALITY NOTICE: This e-mail message and any
attachme...{{dropped}}
Hi,
I get your point with interpreting absent/present calls. Technically
it's a
nice feature, becasue one can just discard the majority of the genes
on the
chip for further analysis. In fact I think absent/present calls make
sense in
terms of biology, since just a fraction of the genes are realy
expressed at a
time. How to express this numerially is a different story (and I guess
a
difficult one).
Anyway, with MAS the calls are calculated anyway, can't they? So, I'd
be nice
(at least for "completness") to add a "mascall" method to the exprSet
objects
generated by affy. What do you think?
By the way, if you ignore the call, do you set an arbitrary intensity
cutoff
later in your analysis, or do just reley on the statistics (anova
p-value or
whatever)?
regards,
Arne
> -----Original Message-----
> From: A.J. Rossini [mailto:rossini@blindglobe.net]
> Sent: 08 October 2003 15:33
> To: Muller, Arne PH/FR
> Cc: bioconductor@stat.math.ethz.ch
> Subject: Re: [BioC] Affy: Present calls in an eset
>
>
> <arne.muller@aventis.com> writes:
>
> > Hello,
> >
> > I'm quite new to Bioconductor/affy, and I was wondering if
> there's a simple
> > way to include the absent/present call for a gene in the
> outputfile generated
> > with write.exprs(eset, file='boo') in theaffy package.
> >
> > the eset was generated with
> >
> > eset <- expresso(cel, bgcorrect.method = 'rma',
> normalize.method =
> > 'qspline', pmcorrect.method = 'pmonly',
> > summary.method='liwong')
> >
> > For further analyses I'd like to exclude genes that are
> absent in all chips.
>
> That's tough. It isn't clear what a sensible definition of absent
> is. Or present.
>
> Do you mean "expressed" ? "Differentially expressed" ? "sort of
> differentially expressed but not too weakly expressed?". For any of
> these, you'll need a precise definition (there isn't any in
> Bioconductor), and you can compute your own.
>
> (I know that MAS will make these calls; I'm only familiar with
Rosetta
> Resolver's variant, and they don't really make sense to me -- to be
> precise, I know numerically how they are derived, but fail to why
they
> realistically connect biologically or technologically without a
great
> deal of assumptions and a wild imagination).
>
> best,
> -tony
>
> --
> rossini@u.washington.edu
> http://www.analytics.washington.edu/
> Biomedical and Health Informatics University of Washington
> Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer
> Research Center
> UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is
unreliable
> FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
>
> CONFIDENTIALITY NOTICE: This e-mail message and any attachments may
be
> confidential and privileged. If you received this message in error,
> please destroy it and notify the sender. Thank you.
>
we will *try* to have mascall fro the next release.
On Wed, 8 Oct 2003 Arne.Muller@aventis.com wrote:
> Hi,
>
> I get your point with interpreting absent/present calls. Technically
it's a
> nice feature, becasue one can just discard the majority of the genes
on the
> chip for further analysis. In fact I think absent/present calls make
sense in
> terms of biology, since just a fraction of the genes are realy
expressed at a
> time. How to express this numerially is a different story (and I
guess a
> difficult one).
>
> Anyway, with MAS the calls are calculated anyway, can't they? So,
I'd be nice
> (at least for "completness") to add a "mascall" method to the
exprSet objects
> generated by affy. What do you think?
>
> By the way, if you ignore the call, do you set an arbitrary
intensity cutoff
> later in your analysis, or do just reley on the statistics (anova
p-value or
> whatever)?
>
> regards,
>
> Arne
>
>
> > -----Original Message-----
> > From: A.J. Rossini [mailto:rossini@blindglobe.net]
> > Sent: 08 October 2003 15:33
> > To: Muller, Arne PH/FR
> > Cc: bioconductor@stat.math.ethz.ch
> > Subject: Re: [BioC] Affy: Present calls in an eset
> >
> >
> > <arne.muller@aventis.com> writes:
> >
> > > Hello,
> > >
> > > I'm quite new to Bioconductor/affy, and I was wondering if
> > there's a simple
> > > way to include the absent/present call for a gene in the
> > outputfile generated
> > > with write.exprs(eset, file='boo') in theaffy package.
> > >
> > > the eset was generated with
> > >
> > > eset <- expresso(cel, bgcorrect.method = 'rma',
> > normalize.method =
> > > 'qspline', pmcorrect.method = 'pmonly',
> > > summary.method='liwong')
> > >
> > > For further analyses I'd like to exclude genes that are
> > absent in all chips.
> >
> > That's tough. It isn't clear what a sensible definition of absent
> > is. Or present.
> >
> > Do you mean "expressed" ? "Differentially expressed" ? "sort of
> > differentially expressed but not too weakly expressed?". For any
of
> > these, you'll need a precise definition (there isn't any in
> > Bioconductor), and you can compute your own.
> >
> > (I know that MAS will make these calls; I'm only familiar with
Rosetta
> > Resolver's variant, and they don't really make sense to me -- to
be
> > precise, I know numerically how they are derived, but fail to why
they
> > realistically connect biologically or technologically without a
great
> > deal of assumptions and a wild imagination).
> >
> > best,
> > -tony
> >
> > --
> > rossini@u.washington.edu
> > http://www.analytics.washington.edu/
> > Biomedical and Health Informatics University of Washington
> > Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer
> > Research Center
> > UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is
unreliable
> > FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
> >
> > CONFIDENTIALITY NOTICE: This e-mail message and any attachments
may be
> > confidential and privileged. If you received this message in
error,
> > please destroy it and notify the sender. Thank you.
> >
>
> _______________________________________________
> Bioconductor mailing list
> Bioconductor@stat.math.ethz.ch
> https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
>
fyi,
we just put into the devel version of affy the method "mas5calls",
that
works on instance of AffyBatch and ProbeSet, that for AffyBatch
returns an
exprSet with MAS 5.0 P,M,A calls in the exprs slot and the wilcoxon
test pvalue in the se.exprs slot. According to our tests, The P,M,A
calls
reproduce affymetrix's almost perfectly. the p-values dont match up as
well.
we would greatly appreciate feedback.
the engine function mas5.detection that works on nx2 matrices, was
contributed by Ben Rubinstein
-r
On Wed, 8 Oct 2003, Rafael A. Irizarry wrote:
> we will *try* to have mascall fro the next release.
>
> On Wed, 8 Oct 2003 Arne.Muller@aventis.com wrote:
>
> > Hi,
> >
> > I get your point with interpreting absent/present calls.
Technically it's a
> > nice feature, becasue one can just discard the majority of the
genes on the
> > chip for further analysis. In fact I think absent/present calls
make sense in
> > terms of biology, since just a fraction of the genes are realy
expressed at a
> > time. How to express this numerially is a different story (and I
guess a
> > difficult one).
> >
> > Anyway, with MAS the calls are calculated anyway, can't they? So,
I'd be nice
> > (at least for "completness") to add a "mascall" method to the
exprSet objects
> > generated by affy. What do you think?
> >
> > By the way, if you ignore the call, do you set an arbitrary
intensity cutoff
> > later in your analysis, or do just reley on the statistics (anova
p-value or
> > whatever)?
> >
> > regards,
> >
> > Arne
> >
> >
> > > -----Original Message-----
> > > From: A.J. Rossini [mailto:rossini@blindglobe.net]
> > > Sent: 08 October 2003 15:33
> > > To: Muller, Arne PH/FR
> > > Cc: bioconductor@stat.math.ethz.ch
> > > Subject: Re: [BioC] Affy: Present calls in an eset
> > >
> > >
> > > <arne.muller@aventis.com> writes:
> > >
> > > > Hello,
> > > >
> > > > I'm quite new to Bioconductor/affy, and I was wondering if
> > > there's a simple
> > > > way to include the absent/present call for a gene in the
> > > outputfile generated
> > > > with write.exprs(eset, file='boo') in theaffy package.
> > > >
> > > > the eset was generated with
> > > >
> > > > eset <- expresso(cel, bgcorrect.method = 'rma',
> > > normalize.method =
> > > > 'qspline', pmcorrect.method = 'pmonly',
> > > > summary.method='liwong')
> > > >
> > > > For further analyses I'd like to exclude genes that are
> > > absent in all chips.
> > >
> > > That's tough. It isn't clear what a sensible definition of
absent
> > > is. Or present.
> > >
> > > Do you mean "expressed" ? "Differentially expressed" ? "sort
of
> > > differentially expressed but not too weakly expressed?". For
any of
> > > these, you'll need a precise definition (there isn't any in
> > > Bioconductor), and you can compute your own.
> > >
> > > (I know that MAS will make these calls; I'm only familiar with
Rosetta
> > > Resolver's variant, and they don't really make sense to me -- to
be
> > > precise, I know numerically how they are derived, but fail to
why they
> > > realistically connect biologically or technologically without a
great
> > > deal of assumptions and a wild imagination).
> > >
> > > best,
> > > -tony
> > >
> > > --
> > > rossini@u.washington.edu
> > > http://www.analytics.washington.edu/
> > > Biomedical and Health Informatics University of Washington
> > > Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer
> > > Research Center
> > > UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is
unreliable
> > > FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
> > >
> > > CONFIDENTIALITY NOTICE: This e-mail message and any attachments
may be
> > > confidential and privileged. If you received this message in
error,
> > > please destroy it and notify the sender. Thank you.
> > >
> >
> > _______________________________________________
> > Bioconductor mailing list
> > Bioconductor@stat.math.ethz.ch
> > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
> >
>
>
--
+---------------------------------------------------------------------
--+
| Rafael Irizarry phone: (410) 614-5157
|
| Assistant Professor fax: (410) 955-0958
|
| Department of Biostatistics office: E3035
|
| Johns Hopkins University email: rafa@jhu.edu
|
+---------------------------------------------------------------------
--+
Hi,
my understading ot the A/P calls from Affy MAS5 is that they represent
a way
to "evaluate" the background signal.
In that way you could have a high background, ie, a high MisM signal,
and
despite the PM signal is identical, for instance 500, the this gene is
going to
be considered as Absent, which could be that your gene is not
expressed, but
could also be due to the design of the probe set rather than to the
fact that
the gene is not expressed....
Detecting if a gene is expressed or not with Affy chips is for me
almost
another question, a difficult one ! my threshold is 70 (2^RMA value)
but it is
likely that at this value some genes are expressed it is hust that
below tis
value I consider, arbitrarly, that it is difficult to validate
something because
of the noise.
Anybody can correct me if I m wrong with all this since I still new
in ths
field as well !
Hope it helps
Philippe
[[alternative HTML version deleted]]
<arne.muller@aventis.com> writes:
> I get your point with interpreting absent/present calls. Technically
it's a
> nice feature, becasue one can just discard the majority of the genes
on the
> chip for further analysis. In fact I think absent/present calls make
sense in
> terms of biology, since just a fraction of the genes are realy
expressed at a
> time. How to express this numerially is a different story (and I
guess a
> difficult one).
Sure, but equivalently, I could implement the "feature" with "Tony's
Complete Crud Calls" (TCCC):
rbinom(40000,1,0.2)
Change the 20% expressed to a different number if it makes you feel
better.
I'm not being quite fair, but from my perspective, it's only slightly
less realistic than the MAS or Resolver calls -- the difference being
that the probability of response is an unclear function of the
expression level, with a trend where the probabilty of the call goes
up with the expression level (which is the technical component).
And then, you've got the possibility of low levels of expression which
are real but hard to pick out.
> Anyway, with MAS the calls are calculated anyway, can't they? So,
I'd be nice
> (at least for "completness") to add a "mascall" method to the
exprSet objects
> generated by affy. What do you think?
It would be great. As far as I know, the MAS calls are protected as a
trade secret and non-disclosure agreements. Maybe not, but I happen
to know for sure that the rosetta resolver calls are! (or I'd have
implemented them for amusement)
> By the way, if you ignore the call, do you set an arbitrary
intensity cutoff
> later in your analysis, or do just reley on the statistics (anova
p-value or
> whatever)?
If you have biological replicates, then either, depending on what you
are looking for. I've never found an approach which works all the
time. If you have technical replicates, then the calls are necessary,
but note that they don't have biological meaning, just technical
(technology) meaning.
best,
-tony
--
rossini@u.washington.edu
http://www.analytics.washington.edu/
Biomedical and Health Informatics University of Washington
Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer Research
Center
UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is unreliable
FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
CONFIDENTIALITY NOTICE: This e-mail message and any
attachme...{{dropped}}
I too have been looking at this issue and would be interested in any
answer
people might have.
Firstly, my understanding is that it is a detection call ("Is the
transcript
of a particular gene Present or Absent?" -- Affymetrix "Statistical
Algorithms Description Document". This is with reference to the
background
level. The detection call is also referred to under a number of other
pretexts - "discrimination score and probability" for one.
I have been going through the MAS 5 documentation and various other
stuff,
and all I can find to reference how this works, is at the "spot"
level(ncol*nrow position - forgive my naivety but I too started
working in
this field just a month ago and am still coming to terms with
understanding). As to yet I have found nothing as to how to program it
for
an expression set, yet quite obviously this is possible. Stats was
never a
strong point of mine either.
If anyone wants a look, I have written a function to work at the
"spot"
level. It's not at the stage of error checking or anything - it was a
quick
hack.
Thanks,
Julian.
-----Original Message-----
From: bioconductor-bounces@stat.math.ethz.ch
[mailto:bioconductor-bounces@stat.math.ethz.ch] On Behalf Of A.J.
Rossini
Sent: 08 October 2003 15:06
To: Arne.Muller@aventis.com
Cc: bioconductor@stat.math.ethz.ch
Subject: Re: [BioC] Affy: Present calls in an eset
<arne.muller@aventis.com> writes:
> I get your point with interpreting absent/present calls. Technically
it's
a
> nice feature, becasue one can just discard the majority of the genes
on
the
> chip for further analysis. In fact I think absent/present calls make
sense
in
> terms of biology, since just a fraction of the genes are realy
expressed
at a
> time. How to express this numerially is a different story (and I
guess a
> difficult one).
Sure, but equivalently, I could implement the "feature" with "Tony's
Complete Crud Calls" (TCCC):
rbinom(40000,1,0.2)
Change the 20% expressed to a different number if it makes you feel
better.
I'm not being quite fair, but from my perspective, it's only slightly
less realistic than the MAS or Resolver calls -- the difference being
that the probability of response is an unclear function of the
expression level, with a trend where the probabilty of the call goes
up with the expression level (which is the technical component).
And then, you've got the possibility of low levels of expression which
are real but hard to pick out.
> Anyway, with MAS the calls are calculated anyway, can't they? So,
I'd be
nice
> (at least for "completness") to add a "mascall" method to the
exprSet
objects
> generated by affy. What do you think?
It would be great. As far as I know, the MAS calls are protected as a
trade secret and non-disclosure agreements. Maybe not, but I happen
to know for sure that the rosetta resolver calls are! (or I'd have
implemented them for amusement)
> By the way, if you ignore the call, do you set an arbitrary
intensity
cutoff
> later in your analysis, or do just reley on the statistics (anova
p-value
or
> whatever)?
If you have biological replicates, then either, depending on what you
are looking for. I've never found an approach which works all the
time. If you have technical replicates, then the calls are necessary,
but note that they don't have biological meaning, just technical
(technology) meaning.
best,
-tony
--
rossini@u.washington.edu
http://www.analytics.washington.edu/
Biomedical and Health Informatics University of Washington
Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer Research
Center
UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is unreliable
FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
CONFIDENTIALITY NOTICE: This e-mail message and any
attachme...{{dropped}}
_______________________________________________
Bioconductor mailing list
Bioconductor@stat.math.ethz.ch
https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
> > Anyway, with MAS the calls are calculated anyway, can't they? So,
I'd
> be nice
> > (at least for "completness") to add a "mascall" method to the
exprSet
> objects
> > generated by affy. What do you think?
>
>It would be great. As far as I know, the MAS calls are protected as
a
>trade secret and non-disclosure agreements. Maybe not, but I happen
>to know for sure that the rosetta resolver calls are! (or I'd have
>implemented them for amusement)
Actually, Affy is pretty open about their algorithms. See their
"Statistical Algorithms Reference Guide"
http://www.affymetrix.com/support/technical/technotes/statistical_refe
rence_guide.pdf
However, their detection algorithm doesn't seem to tell you exactly
how
they calculate everything. Alternatively, we've been using a paired
t-test
on the raw PM and MM values in a chip (one-sided) and consider genes
to be
expressed/present where PM>MM. I haven't checked to see how our method
compares to Affy's - maybe I will when I get some free time :)
Cheers,
Jenny
Jenny Drnevich, Ph.D.
Department of Animal Biology
515 Morrill Hall
505 S Goodwin Ave
Urbana, IL 61801
USA
ph: 217-244-6826
fax: 217-244-4565
e-mail: drnevich@uiuc.edu
Hi,
my understading ot the A/P calls from Affy MAS5 is that they represent
a way
to "evaluate" the background signal.
In that way you could have a high background, ie, a high MisM signal,
and
despite the PM signal is identical, for instance 500, the this gene is
going to
be considered as Absent, which could be that your gene is not
expressed, but
could also be due to the design of the probe set rather than to the
fact that
the gene is not expressed....
Detecting if a gene is expressed or not with Affy chips is for me
almost
another question, a difficult one ! my threshold is 70 (2^RMA value)
but it is
likely that at this value some genes are expressed it is hust that
below tis
value I consider, arbitrarly, that it is difficult to validate
something because
of the noise.
Anybody can correct me if I m wrong with all this since I still new
in ths
field as well !
Hope it helps
Philippe
[[alternative HTML version deleted]]
Hi,
MAS5.0 gives you two numbers, the expression level for each gene, and
a
detection p-value that tells you how confident MAS5.0 is that it got
the
expression level right. This p-value is then split into three
categories
(simply by thresholding) to give you Present, Marginal or Absent
calls.
Generally, thresholding by intensity does something similar to using
the
detection p-value, but not always, for every gene, in every situation.
Keeping these two concepts distinct (accuracy and confidence), is IMHO
not a bad idea (we can of course argue about how best to calculate
metrics for these :-)). After all, this seems to be what we are doing
when we generate not only a fold-change but also a p-score across
replicates for each gene in a study.
Crispin
> -----Original Message-----
> From: Phguardiol@aol.com [mailto:Phguardiol@aol.com]
> Sent: 08 October 2003 15:29
> To: Arne.Muller@aventis.com; bioconductor@stat.math.ethz.ch
> Subject: Re: [BioC] Affy: Present calls in an eset
>
>
> Hi,
> my understading ot the A/P calls from Affy MAS5 is that they
> represent a way
> to "evaluate" the background signal.
> In that way you could have a high background, ie, a high MisM
> signal, and
> despite the PM signal is identical, for instance 500, the
> this gene is going to
> be considered as Absent, which could be that your gene is not
> expressed, but
> could also be due to the design of the probe set rather than
> to the fact that
> the gene is not expressed....
>
> Detecting if a gene is expressed or not with Affy chips is
> for me almost
> another question, a difficult one ! my threshold is 70 (2^RMA
> value) but it is
> likely that at this value some genes are expressed it is hust
> that below tis
> value I consider, arbitrarly, that it is difficult to
> validate something because
> of the noise.
> Anybody can correct me if I m wrong with all this since I
> still new in ths
> field as well !
> Hope it helps
> Philippe
>
> [[alternative HTML version deleted]]
>
> _______________________________________________
> Bioconductor mailing list
> Bioconductor@stat.math.ethz.ch
> https://www.stat.math.ethz.ch/mailman/listinfo> /bioconductor
>
--------------------------------------------------------
This email is confidential and intended solely for the use
o...{{dropped}}
I have a function lying around somewhere, maybe in my AffyExtensions
code
that gives Presence/Absences calls, i think its named callPA() or
something like that. It uses an alternative metric from Affymetrix but
just pretty much does just as well as MAS 5.0 calls (which are pretty
good). of course MAS 5.0 calls, if memory serves me right, are a
little
incompletely describe in the Affymetrix documentation. But It is not
difficult to come up with an alternative method that does pretty well.
Also, Ben Rubenstein gave a recent talk on the issue of detection. You
can
download it from the Affy Low level workshop page
http://www.affymetrix.com/corporate/events/seminar/microarray_workshop
.affx
That's great news, Rafael! Thanks!
The absent/present calls are already included in the TXT files from
the Affy
software (based on MAS). I was wondering if it's possible to getse
files into
the Bioconductor affy package. This's be some kind of high level
analysis,
since the "summary" per gene (the intensity) is already calculated by
the
Affymetrix software based on PM/MM.
As understand the Bioconductor affy package, the normalisation is done
one
the probeset level (for each of the 40 oligos per gene), rather than
the
gene's intensity (affy normalisation is done *before* the probesets
are
summarised into genes). This is somehow different of what we (and
others) do
- although I guess it's a good idea to perform normalisation at the
"lowest"
end, ie he probesets. Is it possible to apply the "normalize.methods"
to
background + pm corrected and summarised genes; i.e.
1. read probe level data
2. background corredtion
3. probe specific bg correction (e.g. substracting MM)
4. Summarizing the probe set values into one expression value
5. normalize summarized expression values
kind regards,
Arne
> -----Original Message-----
> From: Rafael A. Irizarry [mailto:ririzarr@jhsph.edu]
> Sent: 08 October 2003 16:54
> To: Muller, Arne PH/FR
> Cc: rossini@u.washington.edu; bioconductor@stat.math.ethz.ch
> Subject: RE: [BioC] Affy: Present calls in an eset
>
>
> we will *try* to have mascall fro the next release.
>
> On Wed, 8 Oct 2003 Arne.Muller@aventis.com wrote:
>
> > Hi,
> >
> > I get your point with interpreting absent/present calls.
> Technically it's a
> > nice feature, becasue one can just discard the majority of
> the genes on the
> > chip for further analysis. In fact I think absent/present
> calls make sense in
> > terms of biology, since just a fraction of the genes are
> realy expressed at a
> > time. How to express this numerially is a different story
> (and I guess a
> > difficult one).
> >
> > Anyway, with MAS the calls are calculated anyway, can't
> they? So, I'd be nice
> > (at least for "completness") to add a "mascall" method to
> the exprSet objects
> > generated by affy. What do you think?
> >
> > By the way, if you ignore the call, do you set an arbitrary
> intensity cutoff
> > later in your analysis, or do just reley on the statistics
> (anova p-value or
> > whatever)?
> >
> > regards,
> >
> > Arne
> >
> >
> > > -----Original Message-----
> > > From: A.J. Rossini [mailto:rossini@blindglobe.net]
> > > Sent: 08 October 2003 15:33
> > > To: Muller, Arne PH/FR
> > > Cc: bioconductor@stat.math.ethz.ch
> > > Subject: Re: [BioC] Affy: Present calls in an eset
> > >
> > >
> > > <arne.muller@aventis.com> writes:
> > >
> > > > Hello,
> > > >
> > > > I'm quite new to Bioconductor/affy, and I was wondering if
> > > there's a simple
> > > > way to include the absent/present call for a gene in the
> > > outputfile generated
> > > > with write.exprs(eset, file='boo') in theaffy package.
> > > >
> > > > the eset was generated with
> > > >
> > > > eset <- expresso(cel, bgcorrect.method = 'rma',
> > > normalize.method =
> > > > 'qspline', pmcorrect.method = 'pmonly',
> > > > summary.method='liwong')
> > > >
> > > > For further analyses I'd like to exclude genes that are
> > > absent in all chips.
> > >
> > > That's tough. It isn't clear what a sensible definition of
absent
> > > is. Or present.
> > >
> > > Do you mean "expressed" ? "Differentially expressed" ? "sort
of
> > > differentially expressed but not too weakly expressed?".
> For any of
> > > these, you'll need a precise definition (there isn't any in
> > > Bioconductor), and you can compute your own.
> > >
> > > (I know that MAS will make these calls; I'm only familiar
> with Rosetta
> > > Resolver's variant, and they don't really make sense to
> me -- to be
> > > precise, I know numerically how they are derived, but
> fail to why they
> > > realistically connect biologically or technologically
> without a great
> > > deal of assumptions and a wild imagination).
> > >
> > > best,
> > > -tony
> > >
> > > --
> > > rossini@u.washington.edu
> > > http://www.analytics.washington.edu/
> > > Biomedical and Health Informatics University of Washington
> > > Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer
> > > Research Center
> > > UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail
> is unreliable
> > > FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
> > >
> > > CONFIDENTIALITY NOTICE: This e-mail message and any
> attachments may be
> > > confidential and privileged. If you received this message
> in error,
> > > please destroy it and notify the sender. Thank you.
> > >
> >
> > _______________________________________________
> > Bioconductor mailing list
> > Bioconductor@stat.math.ethz.ch
> > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
> >
>
>
I actually asked about this a while back:
https://www.stat.math.ethz.ch/pipermail/bioconductor/2003-August/00208
1.html
The algorithms aren't too hard to code, although I haven't found the
time to
try it yet.
Overall, I don't think the calls are particularly valuable: as Tony
says, if
you read the algorithm you start to wonder at how arbitrary it all is.
On the
other hand, I agree that it would be nice to be replicate this part of
MAS5
with Affy.
Paul
-----Original Message-----
Date: Wed, 8 Oct 2003 15:47:42 +0200
From: <arne.muller@aventis.com>
Subject: RE: [BioC] Affy: Present calls in an eset
To: <rossini@u.washington.edu>
Cc: bioconductor@stat.math.ethz.ch
Message-ID:
<c80ecafa2acc1b45be45d133ed660ade410b0e@crbsmxsusr04.pharma.aventis.co m="">
Content-Type: text/plain; charset="iso-8859-1"
Hi,
I get your point with interpreting absent/present calls. Technically
it's a
nice feature, becasue one can just discard the majority of the genes
on the
chip for further analysis. In fact I think absent/present calls make
sense in
terms of biology, since just a fraction of the genes are realy
expressed at a
time. How to express this numerially is a different story (and I guess
a
difficult one).
Anyway, with MAS the calls are calculated anyway, can't they? So, I'd
be nice
(at least for "completness") to add a "mascall" method to the exprSet
objects
generated by affy. What do you think?
By the way, if you ignore the call, do you set an arbitrary intensity
cutoff
later in your analysis, or do just reley on the statistics (anova
p-value or
whatever)?
regards,
Arne
> -----Original Message-----
> From: A.J. Rossini [mailto:rossini@blindglobe.net]
> Sent: 08 October 2003 15:33
> To: Muller, Arne PH/FR
> Cc: bioconductor@stat.math.ethz.ch
> Subject: Re: [BioC] Affy: Present calls in an eset
>
>
> <arne.muller@aventis.com> writes:
>
> > Hello,
> >
> > I'm quite new to Bioconductor/affy, and I was wondering if
> there's a simple
> > way to include the absent/present call for a gene in the
> outputfile generated
> > with write.exprs(eset, file='boo') in theaffy package.
> >
> > the eset was generated with
> >
> > eset <- expresso(cel, bgcorrect.method = 'rma',
> normalize.method =
> > 'qspline', pmcorrect.method = 'pmonly',
> > summary.method='liwong')
> >
> > For further analyses I'd like to exclude genes that are
> absent in all chips.
>
> That's tough. It isn't clear what a sensible definition of absent
> is. Or present.
>
> Do you mean "expressed" ? "Differentially expressed" ? "sort of
> differentially expressed but not too weakly expressed?". For any of
> these, you'll need a precise definition (there isn't any in
> Bioconductor), and you can compute your own.
>
> (I know that MAS will make these calls; I'm only familiar with
Rosetta
> Resolver's variant, and they don't really make sense to me -- to be
> precise, I know numerically how they are derived, but fail to why
they
> realistically connect biologically or technologically without a
great
> deal of assumptions and a wild imagination).
>
> best,
> -tony
>
> --
> rossini@u.washington.edu
> http://www.analytics.washington.edu/
> Biomedical and Health Informatics University of Washington
> Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer
> Research Center
> UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is
unreliable
> FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email
>
> CONFIDENTIALITY NOTICE: This e-mail message and any attachments may
be
> confidential and privileged. If you received this message in error,
> please destroy it and notify the sender. Thank you.
>
Hi, it's probably also worth remembering that %present calls are
widely
used by the Affy community as part of their standard QC proceedure to
check that arrays within a project are performing sensibly.
Crispin
--------------------------------------------------------
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Indeed %present calls is arguably the best of all data quality
indicators that are suggested by Affymetrix. If you rehybe the same
hybe mix to chips under different conditions - change scanner,
hybridization time or temperature, hybe station - %present calls can
vary widely. Genes don't appear and disappear out of the hybe mix,
but probe affinities change under the different conditions. Making
sure that %present calls are consistent across a set of chips is a way
to check that the processing and experimental conditions that affect
hybridization kinetics were fairly consistent across a set of chips.
As for the Present calls ability to discriminate between samples in
which a given mRNA fragment is present vs a samples in which it isn't,
it will vary from probe set to probe set. In an ideal probe sets in
which all PM/MM probe pairs have similar non-specific binding
affinities and the PM probe has good binding affinity to the target
mRNA fragment, and the target doesn't bind to too many other probes on
the chip, the calls will work well. It is not clear for what
proportion of probe sets the calls actually work as intended. You can
definitely find probe sets for which MM>>PM for several probe pairs in
the set and these fragments will never be called present. The reverse
is also true.
Very little has been published on the subject as far as I know. There
is the work by Ben Rubenstein mentioned earlier in this thread. More
work obviously need to go into this question. I think that one should
be aware that by screening out absent calls, you may be losing many
interesting target fragments. In the days of MAS 4.0, I recall some
genes with negative expression being very good discriminators of tumor
class.
francois
[[alternative HTML version deleted]]
As regards calculating P and A calls like MAS5--I would have imagined
that
this has all the problems inherent in calculating MAS5 values. The
gcrma
algorithm appears to do a better job of calculating real background
from
pooling MM probes of similar gc content. I would have thought this
would be
best for P and A calls too???
Stephen Henderson
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