Mar 2002 (GG) 1

Clustering Gene Expression Data

• Gene Expression Data• Clustering of Genes and Conditions• Methods

– Agglomerative Hierarchical: Average Linkage

– Centroids: K-Means

– Physically motivated: Super-Paramagnetic Clustering

• Coupled Two-Way Clustering

EMBnet: DNA Microarrays Workshop

Mar. 4 – Mar. 8, 2002 ,UNIL & EPFL, Lausanne

Gaddy Getz, Weizmann Institute, Israel

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Gene Expression Technologies

• DNA Chips (Affymetrix) and MicroArrays can measure mRNA concentration of thousands of genes simultaneously

• General scheme: Extract RNA, synthesize labeled cDNA, Hybridize with DNA on chip.

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Single Experiment

• After hybridization– Scan the Chip and obtain an image file

– Image Analysis (find spots, measure signal and noise)Tools: ScanAlyze, Affymetrix, …

• Output File– Affymetrix chips: For each gene a reading proportional

to the concentrations and a present/absent call.(Average Difference, Absent Call)

– cDNA MicroArrays: competing hybridization of target and control. For each gene the log ratio of target and control. (CH1I-CH1B, CH2I-CH2B)

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Preprocessing: From one experiment to many

• Chip and Channel Normalization– Aim: bring readings of all experiments to be on the

same scale

– Cause: different RNA amounts, labeling efficiency and image acquisition parameters

– Method: Multiply readings of each array/channel by a scaling factor such that:

• The sum of the scaled readings will be the same for all arrays

• Find scaling factor by a linear fit of the highly expressed genes

– Note: In multi-channel experiments normalize each channel separately.

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Preprocessing: From one experiment to many

• Filtering of Genes– Remove genes that are absent in most

experiments– Remove genes that are constant in all

experiments– Remove genes with low readings which are not

reliable.

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Colon cancer data (Alon et. al.)

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Noise and Repeats

• >90% 2 to 3 fold

• Multiplicative noise

• Repeat experiments

• Log scaledist(4,2)=dist(2,1)

log – log plot

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We can ask many questions?

• Which genes are expressed differently in two known types of conditions?

• What is the minimal set of genes needed to distinguish one type of conditions from the others?

• Which genes behave similarly in the experiments?• How many different types of conditions are there?

Supervised Methods(use predefined labels)

Supervised Methods(use predefined labels)

Unsupervised Methods(use only the data)

Unsupervised Methods(use only the data)

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• Goal A: Find groups of genes that have correlated expression profiles. These genes are believed to belong to the same biological process and/or are co-regulated.

• Goal B: Divide conditions to groups with similar gene expression profiles. Example: divide drugs according to their effect on gene expression.

Unsupervised Analysis

Clustering Methods

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What is clustering?

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T (RESOLUTION)

Cluster Analysis Yields Dendrogram

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What is clustering? More Mathematically

• Input: N data points, Xi, i=1,2,…,N in a D dimensional space. • Goal: Find “natural” groups or clusters.

Data point of same cluster - “more similar”• Tasks:

– Determine number of clusters– Generate a dendrogram– Identify significant “stable” clusters

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Clustering is ill-posed

• Problem specific definitions

• Similarity: which points should be considered close? – Correlation coefficient– Euclidean distance

• Resolution: specify/hierarchical results

• Shape of clusters: general, spherical.

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Similarity Measure

• Similarity measures – Centered Correlation– Uncentered Correlation– Absolute correlation– Euclidean

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Agglomerative Hierarchical Clustering

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Distance between joined clusters

Need to define the distance between thenew cluster and the other clusters.

Single Linkage: distance between closest pair.

Complete Linkage: distance between farthest pair.

Average Linkage: average distance between all pairs

or distance between cluster centers

Need to define the distance between thenew cluster and the other clusters.

Single Linkage: distance between closest pair.

Complete Linkage: distance between farthest pair.

Average Linkage: average distance between all pairs

or distance between cluster centers

Dendrogram

The dendrogram induces a linear ordering of the data points

The dendrogram induces a linear ordering of the data points

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Agglomerative Hierarchical Clustering

• Results depend on distance update method– Single Linkage: elongated clusters– Complete Linkage: sphere-like clusters

• Greedy iterative process

• NOT robust against noise

• No inherent measure to choose the clusters

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Centroid Methods - K-means

Iteration = 0

•Start with random position of K centroids.

•Iteratre until centroids are stable

•Assign points to centroids

•Move centroids to centerof assign points

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•Start with random position of K centroids.

•Iteratre until centroids are stable

•Assign points to centroids

•Move centroids to centerof assign points

Iteration = 1

Centroid Methods - K-means

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•Start with random position of K centroids.

•Iteratre until centroids are stable

•Assign points to centroids

•Move centroids to centerof assign points

Iteration = 1

Centroid Methods - K-means

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Iteration = 3

•Start with random position of K centroids.

•Iteratre until centroids are stable

•Assign points to centroids

•Move centroids to centerof assign points

Centroid Methods - K-means

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• Result depends on initial centroids’ position

• Fast algorithm: compute distances from data points to centroids

• No way to choose K.

• Example: 3 clusters / K=2, 3, 4

• Breaks long clusters

Centroid Methods - K-means

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Super-Paramagnetic Clustering (SPC) M.Blatt, S.Weisman and E.Domany (1996) Neural Computation

• The idea behind SPC is based on the physical properties dilute magnets.

• Calculating correlation between magnet orientations at different temperatures (T).

T=Low

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• The idea behind SPC is based on the physical properties dilute magnets.

• Calculating correlation between magnet orientations at different temperatures (T).

T=High

Super-Paramagnetic Clustering (SPC) M.Blatt, S.Weisman and E.Domany (1996) Neural Computation

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Super-Paramagnetic Clustering (SPC) M.Blatt, S.Weisman and E.Domany (1996) Neural Computation

• The idea behind SPC is based on the physical properties dilute magnets.

• Calculating correlation between magnet orientations at different temperatures (T).

T=Intermediate

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• The algorithm simulates the magnets behavior at a range of temperatures and calculates their correlation

• The temperature (T) controls the resolution

• Example: N=4800 points in D=2

Super-Paramagnetic Clustering (SPC)

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Output of SPC

Size of largest clusters as function of T

Size of largest clusters as function of T

DendrogramDendrogram

Stable clusters “live” for large T

Stable clusters “live” for large T

A function (T) that peaks when stable clusters break

A function (T) that peaks when stable clusters break

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Choosing a value for T

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Advantages of SPC

• Scans all resolutions (T)

• Robust against noise and initialization -calculates collective correlations.

• Identifies “natural” () and stable clusters (T)

• No need to pre-specify number of clusters

• Clusters can be any shape

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Many clustering methods applied to expression data

• Agglomerative Hierarchical– Average Linkage (Eisen et. al., PNAS 1998)

• Centroid (representative)– K-Means (Golub et. al., Science 1999)

– Self Organized Maps (Tamayo et. al., PNAS 1999)

• Physically motivated – Deterministic Annealing (Alon et. al., PNAS 1999)

– Super-Paramagnetic Clustering (Getz et. al., Physica A 2000)

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Available Tools• Software packages:

– M. Eisen’s programs for clustering and display of results (Cluster, TreeView)

• Predefined set of normalizations and filtering• Agglomerative, K-means, 1D SOM

• Web sites:– Coupled Two-Way Clustering (CTWC) website

http://ctwc.weizmann.ac.il both CTWC and SPC– http://ep.ebi.ac.uk/EP/EPCLUST/

• General mathematical tools– MATLAB

• Agglomerative, public m-files.

– Statistical programs (SPSS, SAS, S-plus)

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Back to gene expression data

• 2 Goals: Cluster Genes and Conditions

• 2 independent clustering:– Genes represented as vectors of expression in

all conditions– Conditions are represented as vectors of

expression of all genes

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Colon cancer data (normalized genes)

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1. Identify tissue classes (tumor/normal)

First clustering - Experiments

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2. Find Differentiating And Correlated Genes

Second Clustering - Genes

Ribosomal proteins Cytochrome C

HLA2

metabolism

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Two-wayClustering

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Coupled Two-Way Clustering (CTWC)G. Getz, E. Levine and E. Domany (2000) PNAS

•Motivation: Only a small subset of genes play a role in

a particular biological process; the other genes

introduce noise, which may mask the signal of the

important players. Only a subset of the samples exhibit

the expression patterns of interest.•New Goal: Use subsets of genes to study subsets of samples (and vice versa) •A non-trivial task – exponential number of subsets.•CTWC is a heuristic to solve this problem.

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Booing

Cheering

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CTWC of colon cancer data

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Glioma cell lineLow grade astrocytomaSecondary GBM

Primary GBMp53 mutation

AB004904 STAT-induced STAT inhibitor 3

M32977 VEGF

M35410 IGFBP2

X51602 VEGFR1

M96322 gravin

AB004903 STAT-induced STAT inhibitor 2

X52946 PTN

J04111 c-jun

X79067 TIS11B

S11S12

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CTWC of Glioblastoma Data – S1(G5)Godard, Getz, Kobayashi, Nozaki, Diserens, Hamon, Stupp, Janzer,

Bucher, de Tribolet, Domany & Hegi (2002) Submitted

AB004904 STAT-induced STAT inhibitor 3M32977 VEGF ANGIOGENESISM35410 IGFBP2 X51602 VEGFR1 ANGIOGENESISM96322 Gravin AB004903 STAT-induced STAT inhibitor 2 X52946 PTNJ04111 C-JUNX79067 TIS11B

AB004904 STAT-induced STAT inhibitor 3M32977 VEGF ANGIOGENESISM35410 IGFBP2 X51602 VEGFR1 ANGIOGENESISM96322 Gravin AB004903 STAT-induced STAT inhibitor 2 X52946 PTNJ04111 C-JUNX79067 TIS11B

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Biological Work

• Literature search for the genes• Genomics: search for common regulatory

signal upstream of the genes • Proteomics: infer functions.• Design next experiment – get more data to

validate result.• Find what is in common with sets of

experiments/conditions.

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Summary

• Clustering methods are used to– find genes from the same biological process

– group the experiments to similar conditions

• Different clustering methods can give different results. The physically motivated ones are more robust.

• Focusing on subsets of the genes and conditions can uncover structure that is masked when using all genes and conditions

http://ctwc.weizmann.ac.il