08-10-2004MCGH Analyzer1 Hans A. Kestler André Müller.
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Transcript of 08-10-2004MCGH Analyzer1 Hans A. Kestler André Müller.
08-10-2004 MCGH Analyzer 2
Data processing steps
• Scanning of the DNA chips (normal and switched)– 2 Channels (Cy 5 and Cy 3)
• Build mean/median over the pixels
• Further processing with MCGH Software
08-10-2004 MCGH Analyzer 3
MCGH software
• Background reductioncalculate intensities according to the background
• Quality control of the spotsreject spots not fitting the quality criteria
• Accumulate spots to clones• Check test
reject clones not fitting the visual options
• Select control clones• Reduce control clones• Main calculation loop
08-10-2004 MCGH Analyzer 5
Background reduction
Background reduction to get intensities1. No reduction2. Fixed reduction3. Local reduction4. Global reduction5. Local + Fixed reduction6. Global + Fixed reduction
Compute log Ratios• log( IntCy3 / IntCy5 )• log( IntCy5 / IntCy3 )
08-10-2004 MCGH Analyzer 6
Quality control
Reject spots with
• flags marked by the scanning software(bad, not found, absent, normal ...)
• A background intensity brighter than the foreground (new!)
• Min/Max reduction:– Reject the n smallest ratios– Reject the n largest ratios
08-10-2004 MCGH Analyzer 7
Spots to clones
Accumulate the non-rejected spot values
• Mean
• Standard deviation
• Median
over
• Intensities (Cy3, Cy5)
• log Ratios
New Feature: Reject clones with less than SpotLowerBound valid spots.
08-10-2004 MCGH Analyzer 8
Check test
Reject clones if at least one of these conditions holds:
1. Me(di)an background intensity > Background upper bound
2. Me(di)an Cy3 Intensity < Me(di)an Cy3 background intensity x Intensity lower bound
3. Standard deviation Cy3 Ratio > Ratio SD upper bound
08-10-2004 MCGH Analyzer 9
Select control clonesOnly non-rejected clones will be selected as control clones.
• Manual selectionSelect clones with id = ‚91‘ or ‚k‘ or ‚K‘ or ‚?91‘ as control clone
• Automatic selection– No [AutoBand]
[CutoffPercentage] clones from the middle band
– [AutoBand]Select band around the median
08-10-2004 MCGH Analyzer 10
Reduce control clonesSome of the control clones will be rejected ...
• [Cutoff Percentage]Reject the n smallest ratios
• Without [Cutoff Band]Reject the n largest ratios
• [Cutoff Band]Reject band around the median
08-10-2004 MCGH Analyzer 11
Main calculation loop1. Calculate control means (the mean/median over all control clones/spots)2. Normalize ratios (subtract control mean from the ratio)3. Calculate tolerance value T
s standard deviation of the ratios of the observed clonen the number of valid spots in this clonet value of the t-statistic
significance niveau
4. [ Force T-Test ]Reject clones with T > [ Force T Value ]
5. [ C Check ]Replace tolerance values with possible greater values.
6. Find clone with maximum tolerance and reject it if its tolerance value T is > [ Force T Value ]
7. Perform [ T Test ] and evaluate result value.
Everything has to be recalculated if a control clone will be rejected.
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08-10-2004 MCGH Analyzer 12
The C CheckThe clone tolerance values are now recalculated according to the following scheme:
If the new tolerance value is greater than the old T will be replaced by the new value
clone in this spots validofnumber
clonecurrent theof ratiosover variance
clonecurrent theof ratiosover mean
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ratiosspot controlover variance
ratiosspot controlover mean
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08-10-2004 MCGH Analyzer 13
The T TestIf [ Force T ] is set, the value will be set to the [ Force T Value ]
otherwise it is the greates tolerance value found in the clones.
clone in this spots validofnumber
clonecurrent theof ratiosover variance
clonecurrent theof ratiosover mean
spots control ofnumber
ratiosspot controlover variance
ratiosspot controlover mean
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08-10-2004 MCGH Analyzer 14
The T Test (2)
• No [ T Test ] : thresholding
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Calculation of the result value R• [ T Test ]
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value thresholdpositive
value thresholdnegative
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In this routine the test T > [ Force T Value ] will be performed repeatedly
08-10-2004 MCGH Analyzer 15
NCBI Clone Database
• Integration of the NCBI “component” database
• Automatically mapping of clone id’s to accession numbers, genomic clone locations and clone status information according to an up-to-date database
• Direct import of the NCBI file format
08-10-2004 MCGH Analyzer 16
Accession-Number
Start-Base End-Base
Clone-State
Database-generated Information
08-10-2004 MCGH Analyzer 17
Batch Processing
•One ore more file pairs can be added to a session
•All computations are performed simultaneous on the included datasets
08-10-2004 MCGH Analyzer 18
Diagrams functions
• Ratio-profiles of multiple clone sets can be shown in one diagram
08-10-2004 MCGH Analyzer 19
Ideogram Browser 1
• Independent portable Java application• Automation from MCGH-Analyzer with JNI• Generation of ideogram drawings from the NCBI
map database• Direct representation of gain and lost markers of
multiple clone sets• Scalable and scrollable graphs
08-10-2004 MCGH Analyzer 21
Software Structure 1
• Excel as convenient platform with widely known user interface for– Table representation– Diagram drawing– User interaction
• Windows DLL written in C++ for high performance using COM automation
• Platform-independent Java-Application for visualizing ideograms (can be docked to the DLL via JNI)