Post on 21-Dec-2015
transcript
HWW Gene Expression Experiments:
How?Why?
What’s the problem?
High Throughput Experiments
Bioinformatics
FunctionalGenomics
DNA Hybridization
• The principle: have two denatured DNA strands bond together, then check double strand amount (florescent dye, radioactive label)
• “Traditional”: Southern/Northern/Western Blot
• The great advance: micro array DNA chips – automation, material eng., computer aided (including algorithmic solutions)
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
HistoryHistory
cDNA microarrays have evolved from Southern blots, with clone libraries gridded out on nylon membrane filters being an important and still widely used intermediate. Things took off with the introduction of non-porous solid supports, such as glass - these permitted miniaturization - and fluorescence based detection. Currently, about 20,000 cDNAs can be spotted onto a microscope slide. The other, Affymetrix technology can produce arrays of 100,000 oligonucleotides on a silicon chip.
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
THE PROCESSTHE PROCESSBuilding the Chip:
MASSIVE PCR PCR PURIFICATION and PREPARATION
PREPARING SLIDES PRINTING
Preparing RNA:
CELL CULTURE AND HARVEST
RNA ISOLATION
cDNA PRODUCTION
Hybing the Chip:POST PROCESSING
ARRAY HYBRIDIZATION
PROBE LABELING
DATA ANALYSIS
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
MASSIVE PCR PCR PURIFICATION and PREPARATION
PREPARING SLIDES
PRINTING
Building the Chip:
Full yeast genome = 6,500 reactions IPA precipitation +EtOH
washes + 384-well format
The arrayer: high precision spotting device capable of printing 10,000 products in 14 hrs, with a plate change every 25 mins
Polylysine coating for adhering PCR products to glass slides
POST PROCESSING
Chemically converting the positive polylysine surface to prevent non-specific hybridization
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
CELL CULTURE AND HARVEST
RNA ISOLATION
cDNA PRODUCTION
Designing experiments to profile conditions/perturbations/mutations and carefully controlled growth conditions
RNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip.
Single strand synthesis or amplification of RNA can be performed. cDNA production includes incorporation of Aminoallyl-dUTP.
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Hybing the Chip:
ARRAY HYBRIDIZATION
PROBE LABELING
DATA ANALYSIS
Cy3 and Cy5 RNA samples are simultaneously hybridized to chip. Hybs are performed for 5-12 hours and then chips are washed.
Two RNA samples are labelled with Cy3 or Cy5 monofunctional dyes via a chemical coupling to AA-dUTP. Samples are purified using a PCR cleanup kit.
Ratio measurements are determined via quantification of 532 nm and 635 nm emission values. Data are uploaded to the appropriate database where statistical and other analyses can then be performed.
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Printing MicroarraysPrinting Microarrays• Print Head• Plate Handling• XYZ positioning
– Repeatability & Accuracy– Resolution
• Environmental Control– Humidity – Dust
• Instrument Control• Sample Tracking Software
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Ngai Lab arrayer , UC Berkeley
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Microarray GridderMicroarray Gridder
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Printing ApproachesPrinting Approaches
Non - Contact • Piezoelectric dispenser• Syringe-solenoid ink-jet dispenser
Contact (using rigid pin tools, similar to filter array)
• Tweezer• Split pin• Micro spotting pin
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Micro Spotting pin
Micro Spotting pin
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Practical ProblemsPractical Problems
—Surface chemistry: uneven surface may lead to high background.
—Dipping the pin into large volume -> pre-printing to drain off excess sample.
—Spot variation can be due to mechanical difference between pins. Pins could be clogged during the printing process.
—Spot size and density depends on surface and solution properties.
—Pins need good washing between samples to prevent sample carryover.
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Post Processing ArraysPost Processing Arrays
Protocol for Post Processing Microarrays
Hydration/Heat Fixing
1. Pick out about 20-30 slides to be processed.
2. Determine the correct orientation of slide, and if necessary, etch label on lower left corner of array side
3. On back of slide, etch two lines above and below center of array to designate array area after processing
4. Pour 100 ml 1X SSC into hydration tray and warm on slide warmer at medium setting
5. Set slide array side down and observe spots until proper hydration is achieved.
6. Upon reaching proper hydration, immediately snap dry slide
7. Place slides in rack.
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Practical Problems 1
• Comet Tails• Likely caused by
insufficiently rapid immersion of the slides in the succinic anhydride blocking solution.
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Practical Problems 2
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Practical Problems 3
High Background• 2 likely causes:
– Insufficient blocking.
– Precipitation of the
labeled probe.
Weak Signals
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Practical Problems 4
Spot overlap:Likely cause: toomuch rehydrationduring post -processing.
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Practical Problems 5
DustDust
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Steps in Images Processing
1. Addressing: locate centers
2. Segmentation: classification of pixels either as signal or background. using seeded region growing).
3. Information extraction: for each spot of the array, calculates signal intensity pairs, background and quality measures.
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Steps in Image Processing
• Spot Intensities– mean (pixel intensities).– median (pixel intensities).
– Pixel variation (IQR of log (pixel
intensities).• Background values
– Local
– Morphological opening
– Constant (global)
– None
• Quality Information
Signal
Background
3. Information Extraction
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Addressing
This is the process of assigning coordinates to each of the spots.
Automating this part of the procedure permits high throughput analysis.
4 by 4 grids19 by 21 spots per grid
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Addressing
Registration
Registration
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Problems in automatic addressing
Misregistration of the red and green channels
Rotation of the array in the image
Skew in the arrayRotation
Rotation
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Segmentation methods• Fixed circles• Adaptive Circle• Adaptive Shape
– Edge detection.– Seeded Region Growing. (R. Adams and L.
Bishof (1994) :Regions grow outwards from the seed points preferentially according to the difference between a pixel’s value and the running mean of values in an adjoining region.
• Histogram Methods– Adaptive threshold.
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Examples of algorithms and software implementation
Methods Software / algorithms
Fixed Circle ScanAlyze, GenePix, QuantArray
Adaptive Circle GenePix
Adaptive Shape Edging and region growing.
Histogram Method QuantArray and adaptivethresholding.
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Limitation of fixed circle method
SRG Fixed Circle
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Limitation of circular segmentation
—Small spot—Not circular
Results from SRG
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Information Extraction
—Spot Intensities—mean (pixel intensities).—median (pixel intensities).
—Background values—Local —Morphological opening—Constant (global)—None
—Quality Information
Take the average
Department of Statistics, University of California, Berkeley , and Division of Genetics and Bioinformatics, Walter and Eliza Hall Institute of Medical Research
Local Backgrounds
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Summary of analysis possibilitiesSummary of analysis possibilitiesDetermine genes which are differentially expressed (this task
can take many forms depending on replication, etc)
Connect differentially expressed genes to sequence databases and perhaps carry out further analyses, e.g. searching for common upstream motifs
Overlay differentially expressed genes on pathway diagrams
Relate expression levels to other information on cells, e.g. known tumour types
Define subclasses (clusters) in sets of samples (e.g. tumours)
Identify temporal or spatial trends in gene expression
Seek roles for genes on the basis of patterns of co-expression
……..much more
Many challenges: transcriptional regulation involves redundancy, feedback, amplification, .. non-linearity
Department of Statistics, University of California, Berkeley, and Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Biological Question
Sample preparationMicroarray
Life Cycle
Data Analysis & Modeling
Microarray Reaction
MicroarrayDetection
Taken from Schena & Davis
Oligonucleotide Arrays
Schadt et al., Journal of Cellular Biochemistry, 2000
Oligonucleotide Arrays Tech.
• ~20 probes per “gene”, 25bases each*
• Probe size: 24x24 micron (contain ~106 copies of the probe)
• Probe is either a Perfect Match (PP) or a Miss Match (MM)
• MM: – usually at the center of the probe– Aim: to give estimate on the random hybrd.
Motivation
• Data is noisy, missing values.• Each array is scanned separately, in different
settings
→ To extract biological meaningful results we need:
1. Good expression estimations
2. Scale/Normalize across arrays
What we need
• Image segmentation• Background/Gradient correction• Artifact detection
• Allow array to array comparison (scale/normalize)• Assess gene presence (quantitative “Measure”)• Find differentially expressed genes
Why isn’t “Normalization” Easy?
• No ability to read mRNA level directly
• Various noise factors → hard to model exactly.
• Variable biological settings, experiment dependent.
• Need to differentiate between changes caused by biological signal from noise artifacts.
Variability Sources
1. Real Biology – 1. Biological noise
2. Biological Signal
2. Sample preparation related
3. Technical dependent
dChip MBEI
• Based on several papers by Li & Wong (PNAS, 2001 vol 98 no.1 and others)
• Implemented on their freely available dChip software
• Model based: The estimation is based on a model of how the probe intensity values respond to changes of the expression levels of the gene
dChip Model
i is the array indexj is the probe index
is the baseline response of the probe due to non specific hybridization
is the additional rate of increase of the PM response
is the rate of increase of the MM response
dChip “Reduced” Model
Basic idea: Least square parameter estimation, iteratively fitting and
dChip “Reduced” Model
For one array, assume that the set has been learned from a large number of arrays, and therefore known and fixed
Given this set, the linear least square estimate for theta is
An approx. Std. can be computed for this estimator:
dChip “Reduced” Model
• Similarly, we regard the set as known, and compute std. for each phi
• We use these estimated Std. to find outlier and exclude them from the computation:
Dchip – Array outliers detection
Dchip – Probe outliers detection
Normalization/Scaling
• We saw how to get MBEI from dchip, i.e measure “quantitation “
• We still need to scale the different arrays:– Arrays usually differ in overall image
brightness (differ in time, place, exper. Cond….)
• This is usually done PRIOR to the “measure quantitation” manipulations (as dChip’s MBEI we just described).
Global – Normalization/Scaling• Suppose we have two arrays X,Y with values
x1…xM and y1 .. yM
• “Global” normalization (MAS 5): find the constant “a” such that
Which means:
When we have multiple arrays then we choose Y to be the avg. of all arrays or compute a such that sum_i (x_i) = constant
Better way: a(x) i.e adopt the fit parameter as a function of expression level ( as by dChip)
dChip – Normalization/Scaling
• Big question: Which gene to use for this scaling??
• There are various ways to choose the set:– “House keeping” genes (Affy. chips)– Spiked controls added in various stages of the
experiment, in a range of concentrations– Both of the above are very good in theory but (still)
not in practice (esp. in Affy chips)– The result: several approaches suggested on how to
use the set of genes tested in the experiments
• We’ll review dChip’s solution: The “Invariant set”
dChip “Invariant Set”
• Main idea:1. Initialize: set of probes P = all probes 2. Order the probes in both arrays by their expression
values3. Give each probe in each array an index according
to it’s relative expression order4. Find a set of probes P’ who’s relative order is similar
in both arrays 5. Set P = P’ and iterate from stage (2) until
convergence6. Use the resulting P to compute a piecewise linear
running median line as the normalization curve
Normalization Tools – Current State• Commonly Used:
– RMA by Speed Lab– dChip by Li & Wong– GeneChip = MAS5 (Affy. built in tool)
• “The Future”:– New Chip design (both Affy. And cDNA) with
better probes, better built in controls etc.– New algorithms – facilitating probes GC content
(gcRMA), location etc.– New MAS tool (this year ?) is also supposed to
incorporate RMA,dChip etc.
How to Measure Performance?
1. Theoretical Validation – use some theoretical assumptions and evaluate Statistical characteristics of the method at hand.
2. Experimental Validation – 1. Use public data sets to measure different aspects of
performance
2. Evaluate relevant characteristics on your data set. Design your data set accordingly (if possible)
A Benchmark for Affy. Expression Measures*
• Main Idea: Define a “universal” test set & test statistics
• Based on 3 publicly available spike in data sets• Tests for:
– Variability across replicate arrays– Response of GE measures to change in abundance of
RNA– Sensitivity of fold change measures to amount of
actual RNA sample– Accuracy of fold change as a measure of relative
expression– Usefulness of raw fold change score to detect
differential expressed genes
* Cope et al. Bioinformatics, 03 (Speed’s Lab)
MA Plot
M1 = X1 – X2
A = (X1 + X2)/ 2 Where Xi is the log2 of expression measure
Variance across replicates plot
Test Statistics: 1. Median std. 2. Avg. R2 (squared corr. coef.) between two replicates
Observed Expression vs. Nominal Expression Plots
Test Statistics: Fit a linear curve and compute1. linear fit slope (should be 1) 2. R2 to the linear fit
ROC Curves
• One of the chief uses of GE arrays is to identify differentially expressed genes
• ROC ( Receiver Operator Characteristic):A graphical representation of both Sens. and Spec. as a function of threshold value
• X axis: TPR (Sens.) • Y axis: FPR (1-Spec.)• In this case: Use fold change as the score,
knowing which probes are spiked or not..
FC ROC PlotsHere actual TP, FP numbers are used for the axes
Test Statistic: AUC (area under the graph)
FC ROC PlotsSame as before, but only for FC = 2 cases (harder)
The Benchmark – Bottom Line• 15 parameters used to test performace
• 3 “synthetic” spike in data sets
• Automatic submission and evaluation tool + comparative results at:www.biostat.jhsph.edu
Other Tests
• Evaluate separately normalization and expression measures techniques ( as by Huffman et al., Genome Biology, Vol. 3, 2002)
• How do we evaluate performance on our own, very specific, data??? ( hint: see next class..)