Dear list,

I think I need to simplify my question.

I have two samples, each from a time point of its embryogenesis. They  
are applied on a two-color Agilent array to compare between each other.

The raw data has a MA-plot like this:

ftp://genome.wustl.edu/private/272205387781472/yong_data.071031/ 
MA_RGLW1.pdf

After "normexp" and global loess, the MA-plot does change it's shape  
as seen here:

ftp://genome.wustl.edu/private/272205387781472/yong_data.071031/ 
MA_MALWC1.pdf

My 1st question:

Does my data have too much differential expression, according to your  
experience?

Apparently, Jianping thinks so.

Then my 2nd question:

Is it still ok to use global loess for normalization?

Thanks so much, I need your opinions.

I am running the latest R and all packages. Commands I used are:

 > RGLWC <- backgroundCorrect(RGLW, method="normexp", offset=50)
 > MALWC <- normalizeWithinArrays(RGLWC, method="loess")


Best,

Yong

On Nov 1, 2007, at 8:47 AM, Jianping Jin wrote:

> Yong,
>
> What is your reference sample(s) for this test run? Looks like the  
> experiment and reference samples are quite different.
>
> JJ-
>
> --On Wednesday, October 31, 2007 4:40 PM -0500 Yong Yin  
> <yyin@watson.wustl.edu> wrote:
>
>> Dear list,
>>
>> I am new to BioConductor, so please forgive me if my questions are
>> naive to you.
>>
>> We designed an Agilent 4x44k array, with the same 44K probes printed
>> 4 times in the 4 blocks. These 44K probes are designed based on a  
>> low-
>> coverage genome sequencing project for a parasitic nematode. Our
>> purpose is to investigate gene expression during early embryogenesis
>> of the nematode.
>>
>> We have received results from a test run to evaluate the array
>> quality. Samples applied on the chip were from two time points during
>> the nematode embryogenesis. As a experiment, I have been following
>> the LIMMA manual step-by-step, treating the results as a simple two-
>> sample comparison with both technical and biological replication. I
>> have uploaded 3 images in the following location and would love to
>> hear what you folks think:
>>
>> ftp://genome.wustl.edu/private/272205387781472/yong_data.071031/
>>
>> The general quality of the array is very good, I can't find any
>> indication of quality problem. The file "MA_RGLW1.pdf" is a MA plot
>> of raw RG data for one of the 4 blocks. After background correction
>> with "normexp" and within-array normalization with global loess, its
>> MA plot is shown as in "MA_MALWC1.pdf".
>>
>> Given that we are studying early embryogenesis, we should expect that
>> a lot of genes are differentially expressed at these two time points.
>> In the MA plots, I think we indeed see lots of DE.  However,
>> according to what I read, the underline assumption for such
>> normalization is that the majority of the genes under investigation
>> should not be differentially expressed. I also read from other
>> people's posts that I should keep the normalization as simple as
>> possible and the "good" data will always be good.
>>
>>  From my MA plots, do you think my normalization is reasonable with
>> this data? If not, are there suggestions what to do? a different
>> normalization method? or even change the design of the array with a
>> set of spike-in control probes to use for normalization?
>>
>> The two time points in this test run are actually the beginning and
>> the ending points of the developmental stages that we are planning to
>> investigate. We are considering to use a pooled-sample as a common
>> reference. We hope a pooled reference like this will decrease the
>> degrees of differential expression between any two samples of our
>> study. Does this sound like a good idea?
>>
>> After normalization with loess, I went ahead to the step of linear
>> modeling with eBayes and got the following QQ plot:
>> "QQPlot_fitLWC2eBayes.pdf'.
>>
>> Does the modeling look reasonable, according to your experience?
>>
>> Any opinions and advices are greatly appreciated.
>>
>> Best,
>>
>> Yong Yin, Ph.D.
>>
>> Senior Scientist
>> Genome Sequencing Center
>> Washington University School of Medicine, Campus box 8501
>> 4444 Forest Park
>> Saint Louis, MO 63108
>>
>> Tel: (314) 286-1415
>>
>>
>>
>> 	[[alternative HTML version deleted]]
>>
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>
>
>
> ##################################
> Jianping Jin Ph.D.
> Bioinformatics scientist
> Center for Bioinformatics
> Room 3133 Bioinformatics building
> CB# 7104
> University of Chapel Hill
> Chapel Hill, NC 27599
> Phone: (919)843-6105
> FAX:   (919)843-3103
> E-Mail: jjin@email.unc.edu
>

Yong Yin, Ph.D.

Senior Scientist
Genome Sequencing Center
Washington University School of Medicine, Campus box 8501
4444 Forest Park
Saint Louis, MO 63108

Tel: (314) 286-1415



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