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Tutorial 8

Clustering

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Clustering• General Methods

– Unsupervised Clustering• Hierarchical clustering• K-means clustering

• Expression data– GEO– UCSC– ArrayExpress

• Tools– EPCLUST– Mev

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Microarray - Reminder

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Expression Data Matrix

• Each column represents all the gene expression levels from a single experiment.

• Each row represents the expression of a gene across all experiments.

Exp1 Exp 2 Exp3 Exp4 Exp5 Exp6

Gene 1 -1.2 -2.1 -3 -1.5 1.8 2.9

Gene 2 2.7 0.2 -1.1 1.6 -2.2 -1.7

Gene 3 -2.5 1.5 -0.1 -1.1 -1 0.1

Gene 4 2.9 2.6 2.5 -2.3 -0.1 -2.3

Gene 5 0.1 2.6 2.2 2.7 -2.1

Gene 6 -2.9 -1.9 -2.4 -0.1 -1.9 2.9

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Expression Data Matrix

Each element is a log ratio: log2 (T/R). T - the gene expression level in the testing sample

R - the gene expression level in the reference sample

Exp1 Exp 2 Exp3 Exp4 Exp5 Exp6

Gene 1 -1.2 -2.1 -3 -1.5 1.8 2.9

Gene 2 2.7 0.2 -1.1 1.6 -2.2 -1.7

Gene 3 -2.5 1.5 -0.1 -1.1 -1 0.1

Gene 4 2.9 2.6 2.5 -2.3 -0.1 -2.3

Gene 5 0.1 2.6 2.2 2.7 -2.1

Gene 6 -2.9 -1.9 -2.4 -0.1 -1.9 2.9

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Microarray Data Matrix

Black indicates a log ratio of zero, i.e.

T=~R

Green indicates a negative log ratio,

i.e. T<R

Red indicates a positive log ratio, i.e. T>R

Grey indicates missing data

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-3

-2

-1

0

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1 2 3 4 5 6

Exp

Log

ratio

Exp

Log

ratio

Microarray Data:Different representations

T<R

T>R

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A real example

~500 genes3 knockdown conditions

To complicate to analyze without “help”

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Microarray Data:Clusters

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How to determine the similarity between two genes? (for clustering)

Patrik D'haeseleer, How does gene expression clustering work?, Nature Biotechnology 23, 1499 - 1501 (2005) , http://www.nature.com/nbt/journal/v23/n12/full/nbt1205-1499.html

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Unsupervised Clustering

Hierarchical Clustering

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genes with similar expression patterns are grouped together and are connected by a series of branches (dendrogram).

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352 4

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35 2 4

Leaves (shapes in our case) represent genes and the length of the paths between leaves represents the distances between genes.

Hierarchical Clustering

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If we want a certain number of clusters we need to cut the tree at a level indicates that number (in this case - four).

Hierarchical clustering finds an entire hierarchy of clusters.

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Hierarchical clustering result

Five clusters

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An algorithm to classify the data into K number of groups.

K=4

K-means Clustering

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How does it work?

The algorithm divides iteratively the genes into K groups and calculates the center of each group. The results are the optimal groups (center distances) for K clusters.

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k initial "means" (in this casek=3) are randomly selected from the data set (shown in color).

k clusters are created by associating every observation with the nearest mean

The centroid of each of the k clusters becomes the new means.

Steps 2 and 3 are repeated until convergence has been reached.

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Different types of clustering – different results

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How to search for expression profiles

• GEO (Gene Expression Omnibus)http://www.ncbi.nlm.nih.gov/geo/

• Human genome browserhttp://genome.ucsc.edu/

• ArrayExpresshttp://www.ebi.ac.uk/arrayexpress/

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Datasets - suitable for analysis with GEO tools

Expression profiles by gene

Microarray experiments

Probe sets

Groups of related microarray experiments

Searching for expression profiles in the GEO

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Download dataset

Clustering

Statistic analysis

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Clustering analysis

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Download dataset

Clustering

Statistic analysis

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The expression distribution for different lines in the cluster

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Searching for expression profiles in the Human Genome browser.

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Keratine 10 is highly expressed

in skin

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http://www.ebi.ac.uk/arrayexpress/

ArrayExpress

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What can we do with all the expression profiles?

Clusters!

How?

EPCLUSThttp://www.bioinf.ebc.ee/EP/EP/EPCLUST/

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Edit the input matrix: Transpose,Normalize,Randomize

Hierarchical clustering

K-means clustering

In the input matrix each column should represents a gene and each row should represent an experiment (or individual).

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Clusters

Data

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Edit the input matrix: Transpose,Normalize,Randomize

Hierarchical clustering

K-means clustering

In the input matrix each column should represents a gene and each row should represent an experiment (or individual).

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Graphical representation of the

cluster

Graphical representation of the

cluster

Samples found in cluster

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10 clusters, as requested

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http://www.tm4.org/mev/

Multi experiment viewer