[BioC] illumina --> limma?

[Stefano Calza]

Thanks Mark for pointing out this.

Regards,
Stefano


On Fri, Aug 25, 2006 at 04:04:47PM +0100, Mark Dunning wrote:
<Mark>So just to clarify, here is the situation regarding BeadArray raw data.
<Mark>
<Mark>If you have made the appropriate tweaks to the BeadScan software then  
<Mark>you should get a text file that look like this (for each array/strip);
<Mark>
<Mark>Code    Grn    GrnX    GrnY
<Mark>50008    796    536.5297    3167.486
<Mark>50008    495    1574.66    12360.35
<Mark>50008    706    2120.334    14611.3
<Mark>50008    519    1493.311    17420.35
<Mark>50008    938    1549.49    11478.03
<Mark>50008    612    1989.498    4735.532
<Mark>50008    744    1073.537    15349.49
<Mark>..........
<Mark>
<Mark>*********************************************
<Mark>For instructions on how to make the tweaks in BeadScan go to
<Mark>
<Mark>http://www.damtp.cam.ac.uk/user/npt22
<Mark>
<Mark>On that page you will find an xml file to replace the existing  
<Mark>Settings.xml file within the BeadScan program directory
<Mark>
<Mark>Note that these steps can only be used before scanning new arrays. I  
<Mark>don't know of any way to recover the x and y coordinates for arrays  
<Mark>that have been previously scanned.
<Mark>
<Mark> *********************************************
<Mark>
<Mark>
<Mark>As Sean notes, each bead is identified by a Code and not a TargetID  
<Mark>as seen in the BeadStudio output. This is annoying, but it is  
<Mark>possible to match up the two identifiers using the bead set manifest  
<Mark>that Illumina distributes on their cd's when they give the arrays   
<Mark>(its a csv file).
<Mark>
<Mark>
<Mark>The Grn column gives the bead intensity that has been calculated  
<Mark>using Illumina image processing methods which include a "sharpening"  
<Mark>transformation and a local background correction. It is possible for  
<Mark>both these steps to produce negative values. The beadarray package is  
<Mark>able to use the TIF images and the X and Y coordinates from the text  
<Mark>file to calculate bead intensities. It is possible to re-create the  
<Mark>steps that Illumina use although we store the local foreground and  
<Mark>background for each bead separately so that background correction is  
<Mark>optional. We are also looking to implement a method of background  
<Mark>estimation using morphological filtering.
<Mark>
<Mark>We have only just seen this new text format for ourselves, so the  
<Mark>code to read these files is only available in the latest version of  
<Mark>beadarray in the developers section.
<Mark>
<Mark>So in summary, with the Code, X and Y information plus the .tif  
<Mark>image, you can re-create the Illumina image analysis yourself to get  
<Mark>raw intensities at the probe level using the beadarray package.
<Mark>Currently this takes around 1min for each array (strip) i.e. about  
<Mark>10-15mins for each chip [ 3Ghz machine with 3GB ram].
<Mark>
<Mark>Hope this helps,
<Mark>
<Mark>
<Mark>
<Mark>Mark
<Mark>
<Mark>On 25 Aug 2006, at 14:55, Stefano Calza wrote:
<Mark>
<Mark>>
<Mark>>
<Mark>>On Fri, Aug 25, 2006 at 09:42:36AM -0400, Sean Davis wrote:
<Mark>><Sean>Mark,
<Mark>><Sean>
<Mark>><Sean>Have you made any headway with Illumina regarding getting  
<Mark>>data that is
<Mark>><Sean>closer to "raw" but still useful?  We are getting the probe- 
<Mark>>level data
<Mark>><Sean>dumped from our (which I think is NOT background-subtracted,  
<Mark>>but correct me
<Mark>><Sean>if I am wrong), but these data do not conform to the usual  
<Mark>>Illumina output
<Mark>><Sean>(different unique ids for the probes), so I really haven't  
<Mark>>given much
<Mark>><Sean>thought to using them, particularly since I can't convince  
<Mark>>our collaborators
<Mark>><Sean>to make hacks of their scanner config file to get the dumps  
<Mark>>for all the data
<Mark>><Sean>we receive.
<Mark>><Sean>
<Mark>>
<Mark>>
<Mark>>I guess we are getting something like that, but afaik the dumps are  
<Mark>>already somehow corrected (and I get some negative values). The  
<Mark>>scanner itself may do some background correction when dumping numbers.
<Mark>>You may need to use the raw scanned image, but it may not be  
<Mark>>possible (if not through an agreement with Illumia).
<Mark>>
<Mark>>I'm just trying to understand what's in this data, so correct me if  
<Mark>>I'm wrong.
<Mark>>
<Mark>>Regards,
<Mark>>Stefano
<Mark>>
<Mark>>-- 
<Mark>>Stefano Calza, PhD
<Mark>>Researcher - Biostatistician
<Mark>>Sezione di Statistica Medica e Biometria
<Mark>>Dipartimento di Scienze Biomediche e Biotecnologie
<Mark>>Università degli Studi di Brescia - Italy
<Mark>>Viale Europa, 11 25123 Brescia
<Mark>>email: calza at med.unibs.it
<Mark>>Phone: +390303717653
<Mark>>Fax: +390303717488
<Mark>>
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<Mark>>Search the archives: http://news.gmane.org/ 
<Mark>>gmane.science.biology.informatics.conductor
<Mark>>

-- 
Stefano Calza, PhD
Researcher - Biostatistician
Sezione di Statistica Medica e Biometria
Dipartimento di Scienze Biomediche e Biotecnologie
Università degli Studi di Brescia - Italy
Viale Europa, 11 25123 Brescia
email: calza at med.unibs.it
Phone: +390303717653
Fax: +390303717488