afgc dna microarray data acquisition and analysis - introduction to stanford microarray database...
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AFGC
DNA Microarray Data Acquisition and
Analysis -
Introduction to Stanford Microarray
Database Damares C MonteCarnegie Institution of Washington,Plant Biology DepartmentStanford, CA
DNA Microarray Technology
Cy3: ~550 nmCy3: ~550 nmCy5: ~650 nmCy5: ~650 nm
No differential expressionNo differential expressionInducedInducedRepressedRepressed
AFGC
• Acquisition - QC
• Input/Storage/Retrieval
• Analysis/Pattern Recognition
• Visualization
• Interpretation/Annotation
• Publication/Repository
SMDSMD
Biologist
Data AnalysisData Analysis
Fluorescence Intensities Extraction
•To extract data from a microarray by accurately identifying the location of each of the spots.
•Data extracted on: Fluorescence intensities, background intensities, fluorescence intensities ratios.
•Load Images
•Adjust Gain (brightness of image) and Normalization (balance between images in red and green)
Create Grids
•Create new grid
•Number of grids to create
•Numbers of rows and columns
•Spot width and height
•Column and Row spacing
•Resolution (Xres and Yres)
•Tip spacing
•Create grid
SMD Overlay Cy3 Cy5
Dust speck
Saturated in both channels
Doughnut
Saturated on outside & in the middle doughnut
Saturated in one channel
Streak
Dust/Background/saturation/something coming into spot
What gets flagged?
Mean Intensity plots
Broad distribution pattern with curved or flattened arms. Common to have gap in the center. (Crab Claw)
Concentrated cluster of spots in a linear or fan pattern with clearly distinguished outliers
Median signal to background (How strong the signal is compared to background)Mean of median background (Determines how high is the background)Median signal to noise (Confidence to quantify peak signal to background) (F635Med - B635Med)/B635SD
Data Analysis FlowData Analysis FlowNew Scan
Gene Pix
Quality Control
Stanford Microarray Database - SMD
Data Selection
Quality Control
Complete Data Table (cdt)
Clustering
DownloadXML
Comparison plotThis plot can be effectively used to get a measure of the overall consistency between two duplicated or reversibly duplicated experiments. It compares the log2(RAT2N) values of the two experiments and calculates the regression line, which optimally should be close to one. Future functions will include filters and plotting of multiple experiments.
Why do we use log space?
Exponential distribution of intensitiesMost genes at low level Very few high level
Log scale approx. normal distribution??
Frequency of intensity levelsThis plot allows the user to plot the frequency of spots within
certain intensity intervals for the red and green channel respectively. It is possible to use normalized or non-normalized intensity values and linear or logarithmic scales. This graph will make it easier to determine background levels for filtering when using other analysis tools. The two examples below give a good
view of what normalization does to the data.
Frequency of intensity levels
Distribution of ratiosThis plot draws the distribution of the logarithm (base 2) of the red/green ratios [log2(RAT2N)]. It gives an immediate overview of the range of expression and the normality of the distribution.
Clustering and Image Generation Page
Spot Data PageSpot Data Page
Things to be Considered When Analyzing Microarray Data
1. Clones corresponding to spots with intensities of at least 350 in both channels and
ratios of 2.0 / 0.5 are worth further investigation.
2. For genes of interest, check that the spot(s) is acceptable.
3. Check for consistency among replicate values.
4. Validate important results obtained from the microarray data by an independent test or method.
@pilot
http://www.stat.berkeley.edu/users/terry/zarray/Html/log.html
AcknowledgementsAcknowledgementsShauna Somerville
Katrina Ramonell Lorne RoseBi-Huei HoSue ThayerShu-Hsing Wu
AcknowledgementsAcknowledgements
Shauna SomervilleShauna Somerville
Bench team
Katrina Ramonell
Lorne RoseBi-Huei HoSue ThayerShu-Hsing Wu
Bioinformatics team
David FinkelsteinJeremy GollubFredrik SterkyRob EwingLalitha SubramanianMira KaloperTodd RichmondMike Cherry
http://www.stat.berkeley.edu/users/terry/zarray/Html/log.html
http://www.stat.berkeley.edu/users/terry/zarray/Html/log.html