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DNA Chips and Their Analysis
Comp. Genomics: Lecture 13based on many sources, primarily Zohar Yakhini
DNA Microarras: Basics
• What are they.• Types of arrays (cDNA arrays, oligo arrays).• What is measured using DNA microarrays. • How are the measurements done?
DNA Microarras: Computational Questions• Design of arrays.• Techniques for analyzing experiments. • Detecting differential expression.• Similar expression: Clustering.• Other analysis techniques (mmmmmany).• Machine learning techniques, and applications
for advanced diagnosis.
What is a DNA Microarray (I)
• A surface (nylon, glass, or plastic).
• Containing hundreds to thousand pixels.
• Each pixel has copies of a sequence
of single stranded DNA (ssDNA).
• Each such sequence is called a probe.
What is a DNA Microarray (II)
• An experiment with 500-10k elements.
• Way to concurrently explore the function of multiple genes.
• A snapshot of the expression level of 500-10k genes under given test conditions
Some Microarray Terminology
• Probe: ssDNA printed on the solid substrate (nylon or glass). These are
short substrings of the genes we are going to be testing
• Target: cDNA which has been labeled and is to be washed over the probe
Back to Basics: Watson and Crick
James Watson and Francis Crick discovered, in 1953, the double helix structure of DNA.
From Zohar Yakhini
Watson-Crick Complimentarity
A binds to TC binds to G
AATGCTTAGTCTTACGAATCAG
Perfect match
AATGCGTAGTCTTACGAATCAG
One-base mismatchFrom Zohar Yakhini
Array Based Hybridization Assays (DNA Chips)
Unknown sequence or mixture (target).Many copies.
• Array of probes• Thousands to millions of
different probe sequences per array.
From Zohar Yakhini
Array Based Hyb Assays
• Target hybs to WC complimentary probes only
• Therefore – the fluorescence pattern is indicative of the target sequence.
From Zohar Yakhini
DNA SequencingSanger Method
• Generate all A,C,G,T – terminated prefixes of the sequence, by a polymerase reaction with terminating corresponding bases.
• Run in four different gel lanes. • Reconstruct sequence from the
information on the lengths of all A,C,G,T – terminated prefixes.
• The need for 4 different reactions is avoided by using differentially dye labeled terminating bases.
From Zohar Yakhini
Central Dogma of Molecular Biology(reminder)
Transcription
mRNA
Cells express different subset of the genes in different tissues and under different conditions
Gene (DNA)
Translation
Protein
From Zohar Yakhini
Expression Profiling on MicroArrays
• Differentially label the query sample and the control (1-3).
• Mix and hybridize to an array.
• Analyze the image to obtain expression levels information.
From Zohar Yakhini
Microarray: 2 Types of Fabrication
1. cDNA Arrays: Deposition of DNA fragments– Deposition of PCR-amplified cDNA clones– Printing of already synthesized oligonucleotieds
2. Oligo Arrays: In Situ synthesis– Photolithography– Ink Jet Printing– Electrochemical Synthesis
By Steve Hookway lecture and Sorin Draghici’s book “Data Analysis Tools for DNA Microarrays”
cDNA Microarrays vs. Oligonucleotide Probes and Cost
cDNA Arrays Oligonucleotide Arrays
•Long Sequences•Spot Unknown Sequences•More variability• Arrays cheaper
•Short Sequences•Spot Known Sequences•More reliable data•Arrays typically more expensive
By Steve Hookway lecture and Sorin Draghici’s book “Data Analysis Tools for DNA Microarrays”
Photolithography (Affymetrix)
• Similar to process used to generate VLSI circuits
• Photolithographic masks are used to add each base
• If base is present, there will be a “hole” in the corresponding mask
• Can create high density arrays, but sequence length is limited
From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Photodeprotection
mask
C
Photolithography (Affymetrix)
From Zohar Yakhini
Ink Jet Printing
• Four cartridges are loaded with the four nucleotides: A, G, C,T
• As the printer head moves across the array, the nucleotides are deposited in pixels where they are needed.
• This way (many copies of) a 20-60 base long oligo is deposited in each pixel.
By Steve Hookway lecture and Sorin Draghici’s book “Data Analysis Tools for DNA Microarrays”
A GTC
…
Ink Jet Printing (Agilent)
The array is a stack of images in the colors A, C, G, T.
From Zohar Yakhini
Inkjet Printed Microarrays
Inkjet head, squirting phosphor-ammodites
From Zohar Yakhini
Electrochemical Synthesis
• Electrodes are embedded in the substrate to manage individual reaction sites
• Electrodes are activated in necessary positions in a predetermined sequence that allows the sequences to be constructed base by base
• Solutions containing specific bases are washed over the substrate while the electrodes are activated
From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Preparation of Samples
• Use oligo(dT) on a separation column to extract mRNA from total cell populations.
• Use olig(dT) initiated polymerase to reverse transcribe RNA into fluorescence labeled cDNA. RNA is unstable because of environment RNA-digesting enzymes.
• Alternatively – use random priming for this purpose, generating a population of transcript subsequences
From Zohar Yakhini
Expression Profiling on MicroArrays
• Differentially label the query sample and the control (1-3).
• Mix and hybridize to an array.
• Analyze the image to obtain expression levels information.
From Zohar Yakhini
Expression Profiling: a FLASH Demo
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
URL:
Expression Profiling – Probe Design Issues
• Probe specificity and sensitivity.• Special designs for splice variations or
other custom purposes.• Flat thermodynamics.• Generic and universal systems
From Zohar Yakhini
Hybridization Probes
• Sensitivity:Strong interaction between the probe and its intended target, under the assay's conditions.How much target is needed for the reaction to be detectable or quantifiable?
• Specificity:No potential cross hybridization.
From Zohar Yakhini
Specificity
• Symbolic specificity
• Statistical protection in the unknown part of the genome.
Methods, software and application in collaboration with Peter Webb, Doron Lipson.
From Zohar Yakhini
Reading Results: Color Coding
• Numeric tables are difficult to read• Data is presented with a color scale• Coding scheme:
– Green = repressed (less mRNA) gene in experiment
– Red = induced (more mRNA) gene in experiment
– Black = no change (1:1 ratio)
• Or– Green = control condition (e.g. aerobic)
– Red = experimental condition (e.g. anaerobic)
• We usually use ratio
Campbell & Heyer, 2003
cDNA array,Inkjet deposition
In-Situ synthesized oligonucleotide array. 25-60 mers.
Thermal Ink Jet Arrays, by Agilent Technologies
Application of Microarrays
• We only know the function of about 30% of the 30,000 genes in the Human Genome– Gene exploration– Functional Genomics
• First among many high
throughput genomic devices
http://www.gene-chips.com/sample1.html
By Steve Hookway lecture and Sorin Draghici’s book “Data Analysis Tools for DNA Microarrays”
A Data Mining Problem
• On a given microarray, we test on the order of 10k elements in one time
• Number of microarrays used in typical experiment is no more than 100.• Insufficient sampling.• Data is obtained faster than it can be processed.• High noise.• Algorithmic approaches to work through this
large data set and make sense of the data are desired.
Informative Genes in aTwo Classes Experiment
• Differentially expressed in the two classes.• Identifying (statistically significant)
informative genes
- Provides biological insight
- Indicate promising research directions
- Reduce data dimensionality
- Diagnostic assay
From Zohar Yakhini
Expression pattern and pathological diagnosis information (annotation), for a single gene
+ + - - + + + - - + - - + + -a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 a11 a12 a13 a14 a15
Permute the annotation by sorting the expression pattern (ascending, say).
Informative genes
+ + + + + + + + - - - - - - -
- - - - - - - + + + + + + + + - - - - + - - + + - + + + + +
etc
Non-informative genes
+ - + - + + + + - - + + - - -
- + + - + - - + + - + + - - + + - - - + + - + + - + + - + -
etc
Scoring Genes
From Zohar Yakhini
Threshold Error Rate (TNoM) Score
Find the threshold that best separates tumors from normals, count the number of errors committed there.
- + + - + - - + + - + + - - +
# of errors = min(7,8) = 7.
6 7
Ex 1:
Ex 2: A perfect single gene classifier gets a score of 0.
+ + + + + + + + - - - - - - -
0 From Zohar Yakhini
p-Values• Relevance scores are more useful when
we can compute their significance:– p-value: The probability of finding a gene with
a given score if the labeling is random
• p-Values allow for higher level statistical assessment of data quality.
• p-Values provide a uniform platform for comparing relevance, across data sets.
• p-Values enable class discovery
From Zohar Yakhini
| | | | | | | - - - - - - - - - - - - - - -
100
200
300
400
500
600
700
Tissues
Ge
ne
s
Breast Cancer BRCA1/ BRCA2 data
BRCA1 Wildtype
Genes over-expressed in BRCA1 wildtype
Genes over-expressed in BRCA1 mutants
BRCA1 mutants
Sporadic sample s14321With BRCA1-mutant expression profile
BRCA1 Differential
Expression
Collab with NIHNEJM 2001
From Zohar Yakhini
Data Analysis: Leave One Out Cross Validation (LOOCV)
Repeat, for each tissue (tumor/normal)
• “Hide” the label of the test tissue• Diagnose the test tissue based on the remaining
data• Compare the diagnosis to the hidden label
Perform this using different choices of genes subsets sizes
Small, efficient diagnostic
assays
From Zohar Yakhini
10
-310
-210
-110
00
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
p-value threshold
Pro
babi
lity
In-silico genotyping of the BRCA1 locus
Correctly classified erroneously classified
BRCA1 LOOCV Results
• 95% success rate (21/22)• Sporadic tissue (14321) consistently classified as BRCA1• BRCA1 gene is normal, but silenced in the patient’s DNA
From Zohar Yakhini
| | | | | | | | | | - - - - - - - - - - - - - - - - - - - - - - - -
50
100
150
200
250
300
350
400
450
500
550
Tissues
Ge
ne
s
LUCA, 38 samples, 14.May.2001 Kaminski.
Lung Cancer Informative Genes
Data from Naftali Kaminski’s lab, at Sheba.
•24 tumors (various types and origins)
•10 normals (normal edges and normal lung pools)
From Zohar Yakhini
And Now: Global Analysisof Gene Expression Data
First (but not least): Clustering
either of genes, or of experiments
Example data: fold change (ratios)
Name 0 hours 2 hours 4 hours 6 hours 8 hours 10 hours
Gene C 1 8 12 16 12 8
Gene D 1 3 4 4 3 2
Gene E 1 4 8 8 8 8
Gene F 1 1 1 0.25 0.25 0.1
Gene G 1 2 3 4 3 2
Gene H 1 0.5 0.33 0.25 0.33 0.5
Gene I 1 4 8 4 1 0.5
Gene J 1 2 1 2 1 2
Gene K 1 1 1 1 3 3
Gene L 1 2 3 4 3 2
Gene M 1 0.33 0.25 0.25 0.33 0.5
Gene N 1 0.125 0.0833 0.0625 0.0833 0.125
Campbell & Heyer, 2003
Example data 2
Name 0 hours 2 hours 4 hours 6 hours 8 hours 10 hours
Gene C 0 3 3.58 4 3.58 3
Gene D 0 1.58 2 2 1.58 1
Gene E 0 2 3 3 3 3
Gene F 0 0 0 -2 -2 -3.32
Gene G 0 1 1.58 2 1.58 1
Gene H 0 -1 -1.60 -2 -1.60 -1
Gene I 0 2 3 2 0 -1
Gene J 0 1 0 1 0 1
Gene K 0 0 0 0 1.58 1.58
Gene L 0 1 1.58 2 1.58 1
Gene M 0 -1.60 -2 -2 -1.60 -1
Gene N 0 -3 -3.59 -4 -3.59 -3
Campbell & Heyer, 2003
Pearson Correlation Coefficient, r. values in [-1,1] interval
• Gene expression over d experiments is a vector in Rd, e.g. for gene C: (0, 3, 3.58, 4, 3.58, 3)
• Given two vectors X and Y that contain N elements, we calculate r as follows:
Cho & Won, 2003
Example: Pearson Correlation Coefficient, r
• X = Gene C = (0, 3.00, 3.58, 4, 3.58, 3)Y = Gene D = (0, 1.58, 2.00, 2, 1.58, 1)
• ∑XY = (0)(0)+(3)(1.58)+(3.58)(2)+(4)(2)+(3.58)(1.58)+(3)(1) = 28.5564
• ∑X = 3+3.58+4+3.58+3 = 17.16• ∑X2 = 32+3.582+42+3.582+32 = 59.6328• ∑Y = 1.58+2+2+1.58+1 = 8.16• ∑Y2 = 1.582+22+22+1.582+12 = 13.9928• N = 6• ∑XY – ∑X∑Y/N = 28.5564 – (17.16)(8.16)/6 = 5.2188 • ∑X2 – (∑X)2/N = 59.6328 – (17.16)2/6 = 10.5552• ∑Y2 – (∑Y)2/N = 13.9928 – (8.16)2/6 = 2.8952• r = 5.2188 / sqrt((10.5552)(2.8952)) = 0.944
Example data: Pearson correlation coefficients
Gene C Gene D Gene E Gene F Gene G Gene H Gene I Gene J Gene K Gene L Gene M Gene N
Gene C 1 0.94 0.96 -0.40 0.95 -0.95 0.41 0.36 0.23 0.95 -0.94 -1
Gene D 0.94 1 0.84 -0.10 0.94 -0.94 0.68 0.24 -0.07 0.94 -1 -0.94
Gene E 0.96 0.84 1 -0.57 0.89 -0.89 0.21 0.30 0.43 0.89 -0.84 -0.96
Gene F -0.40 -0.10 -0.57 1 -0.35 0.35 0.60 -0.43 -0.79 -0.35 0.10 0.40
Gene G 0.95 0.94 0.89 -0.35 1 -1 0.48 0.22 0.11 1 -0.94 -0.95
Gene H -0.95 -0.94 -0.89 0.35 -1 1 -0.48 -0.21 -0.11 -1 0.94 0.95
Gene I 0.41 0.68 0.21 0.60 0.48 -0.48 1 0 -0.75 0.48 -0.68 -0.41
Gene J 0.36 0.24 0.30 -0.43 0.22 -0.21 0 1 0 0.22 -0.24 -0.36
Gene K 0.23 -0.07 0.43 -0.79 0.11 -0.11 -0.75 0 1 0.11 0.07 -0.23
Gene L 0.95 0.94 0.89 -0.35 1 -1 0.48 0.22 0.11 1 -0.94 -0.95
Gene M -0.94 -1 -0.84 0.10 -0.94 0.94 -0.68 -0.24 0.07 -0.94 1 0.94
Gene N -1 -0.94 -0.96 0.40 -0.95 0.95 -0.41 -0.36 -0.23 -0.95 0.94 1Campbell & Heyer, 2003
Example: Reorganization of data
Campbell & Heyer, 2003
Name 0 hours 2 hours 4 hours 6 hours 8 hours 10 hours
Gene M 1 0.33 0.25 0.25 0.33 0.5
Gene N 1 0.125 0.0833 0.0625 0.0833 0.125
Gene H 1 0.5 0.33 0.25 0.33 0.5
Gene K 1 1 1 1 3 3
Gene J 1 2 1 2 1 2
Gene E 1 4 8 8 8 8
Gene C 1 8 12 16 12 8
Gene L 1 2 3 4 3 2
Gene G 1 2 3 4 3 2
Gene D 1 3 4 4 3 2
Gene I 1 4 8 4 1 0.5
Gene F 1 1 1 0.25 0.25 0.1
• Replace each entry xi by its rank in vector x.
• Then compute Pearson correlation coefficients of rank vectors.
• Example: X = Gene C = (0, 3.00, 3.41, 4, 3.58, 3.01) Y = Gene D = (0, 1.51, 2.00, 2.32, 1.58, 1)
• Ranks(X)= (1,2,4,6,5,3)• Ranks(Y)= (1,3,5,6,4,2)• Ties should be taken care of: (1) rare
(2) randomize (small effect)
Spearman Rank Order Coefficient
Grouping and Reduction
• Grouping: Partition items into groups. Items in same group should be similar.
Items in different groups should be dissimilar.
• Grouping may help discover patterns in the data.
• Reduction: reduce the complexity of data by removing redundant probes (genes).
Unsupervised Grouping: Clustering
• Pattern discovery via clustering
similarly expressed genes together
• Techniques most often used:
k-Means Clustering Hierarchical Clustering Biclustering Alternative Methods: Self Organizing Maps (SOMS),
plaid models, singular value decomposition (SVD),
order preserving submatrices (OPSM),……
Clustering Overview
• Different similarity measures in use:– Pearson Correlation Coefficient– Cosine Coefficient– Euclidean Distance– Information Gain– Mutual Information– Signal to noise ratio– Simple Matching for Nominal– –
Clustering Overview (cont.)
• Different Clustering Methods– Unsupervised
• k-means Clustering (k nearest neighbors)• Hierarchical Clustering• Self-organizing map
– Supervised• Support vector machine• Ensemble classifier
Data Mining
Clustering Limitations
• Any data can be clustered, therefore we must be careful what conclusions we draw from our results
• Clustering is often randomized and can and will produce different results for different runs on same data
K-means Clustering
• Given a set of m data points in
n-dimensional space and an integer k.
• We want to find the set of k “centers” in
n-dimensional space that minimizes the Euclidean (mean squared) distance from each data point to its nearest center.
• No exact polynomial-time algorithms are
known for this problem (no wonder, NP-hard!).“A Local Search Approximation Algorithm for k-Means Clustering” by Kanungo
et. al
K-means Heuristic (Lloyd’s Algorithm)
• Has been shown to converge to a locally optimal solution
• But can converge to a solution arbitrarily bad compared to the optimal solution
•“K-means-type algorithms: A generalized convergence theorem and characterization of local optimality” by Selim and Ismail
•“A Local Search Approximation Algorithm for k-Means Clustering” by Kanungo et al.
K=3
Data Points
Optimal Centers
Heuristic Centers
Euclidean Distance
n
iiiE yxyxd
1
2)(),(
543),( 22 AOd E
Now to find the distance between two points, say the origin and the point (3,4):
Simple and Fast! Remember this when we consider the complexity!
Finding a Centroid
We use the following equation to find the n dimensional centroid point (center of mass) amid k (n dimensional) points:
),...,2
,1
(),...,,( 11121 k
xnth
k
ndx
k
stxxxxCP
k
ii
k
ii
k
ii
k
Example: Let’s find the midpoint between three 2D points, say: (2,4) (5,2) (8,9)
2 5 8 4 2 9( , ) (5,5)
3 3CP
K-means Iterative Heuristic
• Choose k initial center points “randomly”• Cluster data using Euclidean distance (or other distance
metric)• Calculate new center points for each cluster, using only points within the cluster• Re-Cluster all data using the new center points
(this step could cause some data points to be placed in a different cluster)
• Repeat steps 3 & 4 until no data points are moved from one cluster to another (stabilization), or till some other convergence criteria is met
From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
An example with 2 clusters
1. We Pick 2 centers at random
2. We cluster our data around these center points
Figure Reproduced From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
K-means example with k=2
3. We recalculate centers based on our current clusters
Figure Reproduced From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
K-means example with k=2
4. We re-cluster our data around our new center points
Figure Reproduced From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
K-means example with k=2
5. We repeat the last two steps until no more data points are moved into a different cluster
Figure Reproduced From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Choosing k
• Run algorithm on data with several different values of k
• Use advance knowledge about the characteristics of your test(e.g. Cancerous vs Non-Cancerous Tissues,
in case the experiments are being clustered)
Cluster Quality
• Since any data can be clustered, how do we know our clusters are meaningful?– The size (diameter) of the cluster
vs. the inter-cluster distance– Distance between the members of a cluster and the
cluster’s center– Diameter of the smallest sphere containing the cluster
From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Cluster Quality Continued
diameter=5
diameter=5distance=2
0
distance=5
Quality of cluster assessed by ratio of distance to nearest cluster and cluster diameter
Figure Reproduced From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Cluster Quality Continued
Quality can be assessed simply by looking at the diameter of a cluster (alone????)
A cluster can be formed by the heuristic even when there is no similarity between clustered patterns. This occurs because the algorithm forces k clusters to be created.From “Data Analysis Tools for DNA Microarrays” by
Sorin Draghici
Characteristics of k-means Clustering
• The random selection of initial center points creates the following properties– Non-Determinism– May produce clusters without patterns
• One solution is to choose the centers randomly from existing patterns
From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Heuristic’s Complexity
• Linear in the number of data points, N
• Can be shown to have run time cN, where c does not depend on N, but rather the number of clusters, k
• (not sure about dependence on dimension, n?)
heuristic is efficient
From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Hierarchical Clustering
- a different clustering paradigm
Figure Reproduced From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
Hierarchical Clustering (cont.)
Gene C Gene D Gene E Gene F Gene G Gene H Gene I Gene J Gene K Gene L Gene M Gene N
Gene C 0.94 0.96 -0.40 0.95 -0.95 0.41 0.36 0.23 0.95 -0.94 -1
Gene D 0.84 -0.10 0.94 -0.94 0.68 0.24 -0.07 0.94 -1 -0.94
Gene E -0.57 0.89 -0.89 0.21 0.30 0.43 0.89 -0.84 -0.96
Gene F -0.35 0.35 0.60 -0.43 -0.79 -0.35 0.10 0.40
Gene G -1 0.48 0.22 0.11 1 -0.94 -0.95
Gene H -0.48 -0.21 -0.11 -1 0.94 0.95
Gene I 0 -0.75 0.48 -0.68 -0.41
Gene J 0 0.22 -0.24 -0.36
Gene K 0.11 0.07 -0.23
Gene L -0.94 -0.95
Gene M 0.94
Gene N
Campbell & Heyer, 2003
Hierarchical Clustering (cont.)
F
C
G
D
E
Gene C Gene D Gene E Gene F Gene G
Gene C 0.94 0.96 -0.40 0.95
Gene D 0.84 -0.10 0.94
Gene E -0.57 0.89
Gene F -0.35
Gene G
C E
1
1 Gene D Gene F Gene G
1 0.89 -0.485 0.92
Gene D -0.10 0.94
Gene F -0.35
Gene G
Average “similarity” to
Gene D: (0.94+0.84)/2 = 0.89
•Gene F: (-0.40+(-0.57))/2 = -0.485
•Gene G: (0.95+0.89)/2 = 0.92
1
Hierarchical Clustering (cont.)
F
G
D
C E
1
1 Gene D Gene F Gene G
1 0.89 -0.485 0.92
Gene D -0.10 0.94
Gene F -0.35
Gene G
G D
2
Hierarchical Clustering (cont.)
F
C E
1
G D
2
1 2 Gene F
1 0.905 -0.485
2 -0.225
Gene F 3
Hierarchical Clustering (cont.)
F C E
1
G D
2
3
3 Gene F
3 -0.355
Gene F
4
F
Hierarchical Clustering (cont.)
F C E
1
G D
2
3
4
algorithm looks familiar?
Clustering of entire yeast genome
Campbell & Heyer, 2003
Hierarchical Clustering:Yeast Gene Expression Data
Eisen et al., 1998
A SOFM Example With Yeast
“Interpresting patterns of gene expression with self-organizing maps: Methods and application to hematopoietic differentiation” by Tamayo et al.
SOM Description
• Each unit of the SOM has a weighted connection to all inputs
• As the algorithm progresses, neighboring units are grouped by similarity
Input Layer
Output Layer
From “Data Analysis Tools for DNA Microarrays” by Sorin Draghici
An Example Using Color
Each color in the map is associated with a weight
From http://davis.wpi.edu/~matt/courses/soms/
Cluster Analysis of Microarray Expression Data Matrices
Application of cluster analysis techniques in the elucidation gene
expression data
The features of a living organism are governed principally by its genes.
If we want to fully understand living systems we must know the function of each gene.
Once we know a gene’s sequence we can design experiments to find its function:
However this approach is too slow to handle all the gene sequence information we have today (HGSP).
Function of Genes
Delete Gene X
Gene X
The Classical Approach of Assigning a function to a Gene
Conclusion: Gene X = left eye gene.
("זבוב בלי רגליים
– חרש")
Microarray AnalysisMicroarray analysis allows the monitoring of the activities of many genes over many different conditions.Experiments are carried out on a Physical Matrix like the one below:
To facilitate computational analysis the physical matrix which may contain 1000’s of gene’s is converted into a numerical matrix using image analysis equipment.
G1G2G3G4G5G6G7G6G7
C1 C2 C3 C4 C5 C6 C7LowZeroHigh
1.55 1.05 0.5 2.5 1.75 0.25 0.1
1.7 0.3 2.4 2.9 1.5 0.5 1.0
1.55 1.05 0.5 2.5 1.75 0.25 0.1
1.7 0.3 2.4 1.5 0.5 1.0
1.55 0.5 2.5 1.75 0.25 0.1
0.3 2.4 2.9 1.5 0.5 1.0
1.55 1.05 0.5 2.5 1.75 0.25 0.1
Conditions
Genes
Possible inference:
If Gene X’s activity (expression) is affected by Condition Y (Extreme Heat), then Gene X may be involved in protecting the cellular components from extreme heat.
Each Gene has its corresponding Expression Profile for a set of conditions.
This Expression Profile may be thought of as a feature profile for that gene for that set of conditions (A condition feature profile).
Cluster Analysis• Cluster Analysis is an unsupervised procedure which involves grouping of objects
based on their similarity in feature space.
• In the Gene Expression context Genes are grouped based on the similarity of their Condition feature profile.
• Cluster analysis was first applied to Gene Expression data from Brewer’s Yeast (Saccharomyces cerevisiae) by Eisen et al. (1998).
Two general conclusions can be drawn from these clusters:
• Genes clustered together may be related within a biological module/system.
• If there are genes of known function within a cluster these may help to class this biological/module system.
X
Y
A
B
C
Z
Clusters A,B and C represent groups of related genes.
Clustering
1.55 1.05 0.5 2.5 1.75 0.25 0.1
1.7 0.3 2.4 2.9 1.5 0.5 1.0
1.55 1.05 0.5 2.5 1.75 0.25 0.1
1.7 0.3 2.4 1.5 0.5 1.0
1.55 0.5 2.5 1.75 0.25 0.1
0.3 2.4 2.9 1.5 0.5 1.0
1.55 1.05 0.5 2.5 1.75 0.25 0.1
Conditions
Genes
From Data to Biological Hypothesis
System C
Cluster C with four Genes may represent System C
Relating these genes aids in elucidation of this System C
Gene Expression Microarray Cluster SetConditions (A-Z)
Gene 1Gene 2Gene 3Gene 4 Gene 5Gene 6Gene 7 X
Y
A
B
C
External Stimulus( Condition X)
Regulator Protein
Toxin
DNA Gene a Gene b Gene c Gene d
Gene Expression
Toxin Pump
Cell Membrane
Some Drawbacks of Clustering Biological Data1. Clustering works well over small numbers of conditions but a typical Microarray
may have hundreds of experimental conditions. A global clustering may not offer sufficient resolution with so many features.
2. As with other clustering applications, it may be difficult to cluster noisy expression data.
3. Biological Systems tend to be inter-related and may share numerous factors (Genes) – Clustering enforces partitions which may not accurately represent these intimacies.
4. Clustering Genes over all Conditions only finds the strongest signals in the dataset as a whole. More ‘local’ signals within the data matrix may be missed.
X
Y
A
B
C
Z
Inter-related(3)
Local Signals(4)
May represent more complex system such as:
How do we better model more complex systems?
• One technique that allows detection of all signals in the data is biclustering.
• Instead of clustering genes over all conditions biclustering clusters genes with respect to subsets of conditions.
-interrelated clusters (genes may belong more than one bicluster).
-local signals (genes correlated over only a few conditions).
-noisy data (allows erratic genes to belong to no cluster).
This enables better representation of:
Biclustering
• Technique first described by J.A. Hartigan in 1972 and termed ‘Direct Clustering’.
• First Introduced to Microarray expression data by Cheng and Church(2000)
Gene 1Gene 2Gene 3Gene 4Gene 5Gene 6Gene 7Gene 8Gene 9
A B C D E F G H
Gene 1
Gene 4
Gene 6
Gene 7
Gene 9
B E F
BiclusteringA B D E F G H
Gene 1
Gene 4
Gene 9
Clustering misses local signal {(B,E,F),(1,4,6,7,9)} present over subset of conditions.
Gene 1
Gene 4
Gene 9
A B C D E F G H
Clustering
Biclustering discovers local coherences over a subset of conditions
Conditions
Approaches to Biclustering Microarray Gene Expression
• First applied to Gene Expression Data by Cheng and Church(2000).– Used a sub-matrix scoring technique to locate biclusters.
• Tanay et al.(2000)– Modelled the expression data on Bipartite graphs and
used graph techniques to find ‘complete graphs’ or biclusters.
• Lazzeroni and Owen– Used matrix reordering to represent different ‘layers’ of
signals (biclusters) ‘Plaid Models’ to represent multiple signals within data.
• Ben-Dor et al. (2002) – “Biclusters” depending on order relations (OPSM).
Bipartite Graph Modelling•First proposed in: “Discovering statically significant biclusters in
gene expressing data” Tanay et al. Bioinformatics 2000
Within the graph modelling paradigm biclusters are equivalent to complete bipartite sub-graphs.
Tanay and colleagues used probabilistic models to determine the least probable sub-graphs (those showing most order and consequently most surprising) to identify biclusters.
1234567
1234567
A B C D E FA
B
C
D
E
F
146
AD146
AD
Graph G
Sub-graph H(Bicluster)
Data Matrix M
Sub-Matrix (Bicluster)
Genes
Genes
Conditions
Conditions
The Cheng and Church Approach
ija
The core element in this approach is the development of a scoring to prioritise sub-matrices.
This scoring is based on the concept of the residue of an entry in a matrix.
In the Matrix (I,J) the residue score of element is given by:
IJIjiJijij aaaaaR )(
ai
jIJ
In words, the residue of an entry is the value of the entry minus the row average, minus the column average, plus the average value in the matrix.
This score gives an idea of how the value fits into the data in the surrounding matrix.
ija
The mean squared residue score (H) for a matrix (I,J) is then calculated :
JjIi
IJIjiJij aaaaJI
JIH,
2)(||||
1),(
This Global H score gives an indication of how the data fits together within that matrix- whether it has some coherence or is random.
The Cheng and Church Approach(2)
A low H score means that there is a correlation in the matrix
- a score of H(I,J)= 0 would mean that the data in the matrix fluctuates in unison i.e. the sub-matrix is a bicluster
A high H value signifies that the data is uncorrelated.
- a matrix of equally spread random values over the range [a,b], has an expected H score of (b-a)/12. range = [0,800] then H(I,J) = 53,333
Worked example of H score:
IJIjiJijij aaaaaR )(
R(1) = 1- 2 - 5.4 + 6.5 = 0.1
R(2) = 2 - 2 - 6.4 + 6.5 = 0.1: :: :
R(12) = 12 - 11 -7.4 + 6.5 = 0.1
Col Avg. 5.4 6.4 7.4
1 2 34 5 67 8 9
10 11 12
Row Avg.
25811
Matrix (M) Avg. = 6.5
H (M) = (0.01x12)/12 = 0.01
If 5 was replaced with 3 then the score would changed to:
H(M2) = 2.06
If the matrix was reshuffled randomly the score would be around:
H(M3) = sqr(12-1)/12 = 10.08
In order to find all possible biclusters in an Expression Matrix all sub-matrices must be tested using the H score.
The Cheng and Church Approach: Node Deletion Biclustering Algorithm
In a node deletion algorithm all columns and rows are tested for deletion. If removing a row or column decreases the H score of the Matrix than it is removed.
This continues until it is not possible to decrease the H score further. This low H score coherent sub-matrix (bicluster) is then returned.
The process then masks this located bicluster by inserting random numbers in place of it.
And reiterates the process.
R
R
R
R
Node Deletion
Node Deletion
The Cheng and Church Approach:
No. of genes, no. of conditions
4, 96 10, 29 11, 25
103, 25 127, 13 13, 21
10, 57 2, 96 25, 12
9, 51 3, 96 2, 96
Some results on lymphoma data (402696):
Conclusions:
• High throughput Functional Genomics (Microarrays) requires Data Mining Applications.
• Biclustering resolves Expression Data more effectively than single dimensional Cluster Analysis.
• Cheng and Church Approach offers good base for future work.
Future Research/Question’s:
• Implement a simple H score program to facilitate study if H score concept.
• Are there other alternative scorings which would better apply to gene expression data?
• Have unbiclustered genes any significance? Horizontally transferred genes?
• Implement full scale biclustering program and look at better adaptation to expression data sets and the biological context.
References
• Basic microarray analysis: grouping and feature reduction by Soumya Raychaudhuri, Patrick D. Sutphin, Jeffery T. Chang and Russ B. Altman; Trends in Biotechnology Vol. 19 No. 5 May 2001
• Self Organizing Maps, Tom Germano, http://davis.wpi.edu/~matt/courses/soms
• “Data Analysis Tools for DNA Microarrays” by Sorin Draghici; Chapman & Hall/CRC 2003
• Self-Organizing-Feature-Maps versus Statistical Clustering Methods: A Benchmark by A. Ultsh, C. Vetter; FG Neuroinformatik & Kunstliche Intelligenz Research Report 0994
References
• Interpreting patterns of gene expression with self-organizing maps: Methods and application to hematopoietic differentiation by Tamayo et al.
• A Local Search Approximation Algorithm for k-Means Clustering by Kanungo et al.
• K-means-type algorithms: A generalized convergence theorem and characterization of local optimality by Selim and Ismail