[BioC] Disorderly duplicate spots
Patrícia Luiza Nunes da Costa
patriciacosta at lim24.fm.usp.br
Fri Jan 18 13:49:36 CET 2008
We used an Affymetrix microarray with about 45 000 genes. We have 4 groups
with 3 arrays.
How many genes should I except after par wise filtering (simpleaffy)? I
know it depends on the parameters and stringency, but I want to know an
average, or the minimum, to perform a statistical analysis.
What algorithm do you think its better: RMA or MAS 5?
Thanks and regards,
> Hi Naomi & Jim,
> thanks for your replies!
> I'll look into doing something along the same lines as you did Naomi.
> Have a wonderful weekend,
> On Jan 17, 2008, at 16:00 , Naomi Altman wrote:
>> Dear Yannick,
>> On the whole, most people have equal numbers of duplicates for each
>> gene, and can use the methods discussed in limma.
>> However, we had a situation similar to yours.
>> First, we did a graphical analysis to determine if the expression
>> profile of a clone set was fairly parallel over the arrays. A
>> parallel profile indicates that the assessment of differential
>> expression will be the same for any clone. (Almost all of ours were,
>> and we suspect that some of the others were possibly assembly
>> errors.) Then we picked the clone that was at a reasonably high
>> quantile of the expression distribution. i.e. we did not pick the
>> most highly expressed clone, in case this was due to some type of
>> error. We picked the median, or the clone at the 75th percentile etc.
>> At 07:48 AM 1/17/2008, Yannick Wurm wrote:
>>> Dear List,
>>> I am a graduate student working with the fire ant Solenopsis invicta.
>>> We did some two-color cDNA microarrays that I've begun analyzing with
>>> limma. But something feels wrong about how I'm doing things: we
>>> printed whole clones from a ~25,000 clone cDNA library onto our
>>> microarray. Simultaneously, we sequenced our clones. They assemble to
>>> ~12,000 transcripts. Many are singlets, but some transcripts are
>>> represented by multiple clones (one transcript is represented by 32
>>> So during analysis, treating each clone as independent feels wrong.
>>> It means:
>>> - correcting for 25,000 multiple tests rather than 10,000,
>>> reducing my power;
>>> - and not taking into account the technical replication we
>>> get by
>>> multiple spots on the array.
>>> The limma manual has a section on Within-Array Replicate Spots. But
>>> only mentions what to do for people who have a single duplicate of
>>> every spot on their array.
>>> I'm sure other people have had to deal with this in the past. Do you
>>> have any pointers?
>>> Thanks & regards,
>>> yannick . wurm @ unil . ch
>>> Ant Genomics, Ecology & Evolution @ Lausanne
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Patrícia Luiza Nunes da Costa
Laboratório de Oncologia Experimental, Grupo de Adesão Celular
Faculdade de Medicina da Universidade de Paulo-FM USP
Av. Dr. Arnaldo, 455 sala 4112
Tel: (11) 3061-7486 e (11) 8202-7073
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