Tutorial 8 Clustering 1
Dec 22, 2015
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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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”
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
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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.
Hierarchical clustering result
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An algorithm to classify the data into K number of groups.
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K=4
K-means Clustering
How does it work?
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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
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Searching for expression profiles in the GEO
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?
EPCLUST
http://www.bioinf.ebc.ee/EP/EP/EPCLUST/
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Edit the input matrix: Transpose,Normalize,Randomize 37
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
Edit the input matrix: Transpose,Normalize,Randomize 39
Hierarchical clustering
K-means clustering
In the input matrix each column should represents a gene and each row should represent an experiment (or individual).
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