Lung Adenocarcinoma: Clustering of mRNA expression: consensus hierarchical
Maintained by Robert Zupko (Broad Institute)
Overview
Introduction

This pipeline calculates clusters based on consensus hierarchical clustering with agglomerative average linkage[1],[2]. This pipeline has the following features:

1. Classify samples into consensus clusters.

2. Determine differentially expressed marker genes for each subtype.

Summary

The 1500 most variable genes were selected. Consensus average linkage hierarchical clustering of 32 samples and 1500 genes identified 3 subtypes with the stability of the clustering increasing for k = 2 to k = 8 and the average silhouette width calculation for selecting the robust clusters.

Results
Silhouette width of each sample in robust cluster

Figure 1. Get High-res Image Silhouette width was calculated and the average silhouette width for all samples within one cluster was shown below according to different clusters (left panel). The robust cluster was pointed out by blue symbol (left panel) and the silhouette width of each sample in robust cluster was shown on right panel.

Gene expression patterns of molecular subtype

Figure 2. Get High-res Image The gene expression heatmap with a standard hierarchical clustering for 32 samples and 1500 most variable genes.

Consensus and correlation matrix

Figure 3. Get High-res Image The consensus matrix after clustering shows 3 robust clusters with limited overlap between clusters.

Figure 4. Get High-res Image The correlation matrix also shows 3 robust clusters.

Samples assignment with silhouette width

Table 1. Get Full Table List of samples with 3 subtypes and silhouette width.

SampleName | cluster | silhouetteValue
--- | --- | ---
TCGA-55-1592-01A-01R-0946-07 | 1 | 0.088
TCGA-55-1594-01A-01R-0946-07 | 1 | 0.31
TCGA-55-1595-01A-01R-0946-07 | 1 | 0.073
TCGA-55-1596-01A-01R-0946-07 | 1 | 0.2
TCGA-64-1677-01A-01R-0946-07 | 1 | 0.12
TCGA-64-1678-01A-01R-0946-07 | 1 | 0.24
TCGA-67-3771-01A-01R-0946-07 | 1 | 0.23
TCGA-35-3615-01A-01R-0946-07 | 2 | 0.13
TCGA-44-2655-01A-01R-0946-07 | 2 | 0.25
TCGA-44-2656-01A-02R-0946-07 | 2 | -0.014

Table 2. Get Full Table List of samples belonging to each cluster in different k clusters.

TCGA_ID | K.2 | K.3 | K.4 | K.5 | K.6 | K.7 | K.8
--- | --- | --- | --- | --- | --- | --- | ---
TCGA-35-3615-01A-01R-0946-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8
TCGA-44-2655-01A-01R-0946-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8
TCGA-44-2656-01A-02R-0946-07 | 1 | 2 | 2 | 5 | 6 | 3 | 5
TCGA-44-2657-01A-01R-1107-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8
TCGA-44-2659-01A-01R-0946-07 | 1 | 2 | 2 | 5 | 6 | 3 | 5
TCGA-44-2661-01A-01R-1107-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8
TCGA-44-2666-01A-01R-0946-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8
TCGA-44-4112-01A-01R-1107-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8
TCGA-64-1680-01A-02R-0946-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8
TCGA-67-3770-01A-01R-0946-07 | 1 | 2 | 2 | 5 | 6 | 5 | 8

Marker genes of each subtype

Samples most representative of the clusters, hereby called core samples were identified based on positive silhouette width, indicating higher similarity to their own class than to any other class member. Core samples were used to select differentially expressed marker genes for each subtype by comparing the subclass versus the other subclasses, using Student's t-test.

Table 3. Get Full Table List of marker genes with p<= 0.05 (The positive value of column difference means gene is upregulated in this subtype and vice versa).

Composite.Element.REF | p | difference | q | subclass
--- | --- | --- | --- | ---
KLK12 | 0.0023 | 4.7 | 0.058 | 1
MAGEA9 | 0.031 | 4.5 | 0.17 | 1
FGL1 | 0.0019 | 4.3 | 0.053 | 1
CTNND2 | 0.0011 | 4.3 | 0.042 | 1
MAGEA4 | 0.02 | 3.9 | 0.14 | 1
FGB | 0.011 | 3.7 | 0.11 | 1
CHRNA9 | 0.022 | 3.7 | 0.15 | 1
MAGEA1 | 0.018 | 3.5 | 0.13 | 1
CSAG3A | 0.028 | 3.5 | 0.16 | 1
ATP6V0A4 | 0.0021 | 3.4 | 0.056 | 1

Methods & Data
Consensus Hierarchical Clustering

Consensus Hierarchical clustering is a resampling-based clustering. It provides for a method to represent the consensus across multiple runs of a clustering algorithm and to assess the stability of the discovered clusters. To this end, perturbations of the original data are simulated by resampling techniques [1],[2].

Silhouette Width

Silhouette width is defined as the ratio of average distance of each sample to samples in the same cluster to the smallest distance to samples not in the same cluster. If silhouette width is close to 1, it means that sample is well clustered. If silhouette width is close to -1, it means that sample is misclassified [3].

Download Results

This is an experimental feature. The full results of the analysis summarized in this report can be downloaded from the TCGA Data Coordination Center.

- Analysis Results (MD5 checksum)

- Auxiliary Data (MD5 checksum)

- MAGE-TAB File (MD5 checksum)

References
[1] Bmonti, S., Tamayo, P., Mesirov, J. & Golub, T.R., Consensus Clustering: A Resampling-Based Method for Class Discovery and Visualization of Gene Expression Microarray Data, Machine Learning:91-118 (2003)
[2] Broad Genepattern: ConsensusClustering
[3] Rousseeuw, P.J., Silhouettes: A graphical aid to the interpretation and validation of cluster analysis., J. Comput. Appl. Math. 20:53-65 (1987)
[4] R silhouette package
Meta

- Maintainer = Robert Zupko

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