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Statistics and Data Analysis for Microarrays Using R and
Bioconductor 2nd Edition Sorin Draghici Digital Instant
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Author(s): Sorin Draghici
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 Computer Science/Bioinformatics

Second
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Richly illustrated in color, Statistics and Data Analysis for

Microarrays Using R and Bioconductor, Second Edition provides

Using R and Bioconductor

for Microarrays
Statistics and Data Analysis
a clear and rigorous description of powerful analysis techniques
and algorithms for mining and interpreting biological information.
Omitting tedious details, heavy formalisms, and cryptic notations,
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control, data pre-processing and normalization, gene ontology
analysis, pathway analysis, and machine learning techniques.
Methods are illustrated with toy examples and real data, and the R
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With all the necessary prerequisites included, this best-selling
book guides readers from very basic notions to advanced analysis
techniques in R and Bioconductor. The first half of the text presents
an overview of microarrays and the statistical elements that form the
building blocks of any data analysis. The second half introduces the
techniques most commonly used in the analysis of microarray data.
Dr ăghici

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 Statistics and Data Analysis
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Using R and Bioconductor
Second Edition

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 CHAPMAN & HALL/CRC
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 Published Titles
Algorithms in Bioinformatics: A Practical Exactly Solvable Models of Biological
Introduction Invasion
Wing-Kin Sung Sergei V. Petrovskii and Bai-Lian Li
Bioinformatics: A Practical Approach Gene Expression Studies Using
Shui Qing Ye Affymetrix Microarrays
Biological Computation Hinrich Göhlmann and Willem Talloen
Ehud Lamm and Ron Unger Glycome Informatics: Methods and
Biological Sequence Analysis Using Applications
the SeqAn C++ Library Kiyoko F. Aoki-Kinoshita
Andreas Gogol-Döring and Knut Reinert Handbook of Hidden Markov Models
Cancer Modelling and Simulation in Bioinformatics
Luigi Preziosi Martin Gollery

Cancer Systems Biology Introduction to Bioinformatics

Edwin Wang Anna Tramontano

Cell Mechanics: From Single Scale- Introduction to Bio-Ontologies

Based Models to Multiscale Modeling Peter N. Robinson and Sebastian Bauer
Arnaud Chauvière, Luigi Preziosi, Introduction to Computational
and Claude Verdier Proteomics
Clustering in Bioinformatics and Drug Golan Yona
Discovery Introduction to Proteins: Structure,
John D. MacCuish and Norah E. MacCuish Function, and Motion
Combinatorial Pattern Matching Amit Kessel and Nir Ben-Tal
Algorithms in Computational Biology An Introduction to Systems Biology:
Using Perl and R Design Principles of Biological Circuits
Gabriel Valiente Uri Alon
Computational Biology: A Statistical Kinetic Modelling in Systems Biology
Mechanics Perspective Oleg Demin and Igor Goryanin
Ralf Blossey Knowledge Discovery in Proteomics
Computational Hydrodynamics of Igor Jurisica and Dennis Wigle
Capsules and Biological Cells Meta-analysis and Combining
C. Pozrikidis Information in Genetics and Genomics
Computational Neuroscience: Rudy Guerra and Darlene R. Goldstein
A Comprehensive Approach Methods in Medical Informatics:
Jianfeng Feng Fundamentals of Healthcare
Data Analysis Tools for DNA Microarrays Programming in Perl, Python, and Ruby
Sorin Draghici Jules J. Berman
Differential Equations and Mathematical Modeling and Simulation of Capsules
Biology, Second Edition and Biological Cells
D.S. Jones, M.J. Plank, and B.D. Sleeman C. Pozrikidis
Dynamics of Biological Systems Niche Modeling: Predictions from
Michael Small Statistical Distributions
Engineering Genetic Circuits David Stockwell
Chris J. Myers

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 Published Titles (continued)
Normal Mode Analysis: Theory and Statistics and Data Analysis for
Applications to Biological and Chemical Microarrays Using R and Bioconductor,
Systems Second Edition
Qiang Cui and Ivet Bahar Sorin Drăghici
Optimal Control Applied to Biological Stochastic Modelling for Systems
Models Biology
Suzanne Lenhart and John T. Workman Darren J. Wilkinson
Pattern Discovery in Bioinformatics: Structural Bioinformatics: An Algorithmic
Theory & Algorithms Approach
Laxmi Parida Forbes J. Burkowski
Python for Bioinformatics The Ten Most Wanted Solutions in
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Spatial Ecology Anna Tramontano
Stephen Cantrell, Chris Cosner, and
Shigui Ruan
Spatiotemporal Patterns in Ecology
and Epidemiology: Theory, Models,
and Simulation
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Ezio Venturino

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 Statistics and Data Analysis
for Microarrays
Using R and Bioconductor
Second Edition

Sorin Drăghici

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CRC Press
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 To Jeannette, my better half,
to Tavi, who brightens every day of my life,
and to Althea, whom I miss every day we are not together
This page intentionally left blank
Contents

List of Figures xxv

List of Tables xxxv

Preface xxxix

1 Introduction 1

1.1 Bioinformatics – an emerging discipline . . . . . . . . . . . . 1

2 The cell and its basic mechanisms 5

2.1 The cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2 The building blocks of genomic information . . . . . . . . . . 13
2.2.1 The deoxyribonucleic acid (DNA) . . . . . . . . . . . 13
2.2.2 The DNA as a language . . . . . . . . . . . . . . . . . 19
2.2.3 Errors in the DNA language . . . . . . . . . . . . . . . 23
2.2.4 Other useful concepts . . . . . . . . . . . . . . . . . . 24
2.3 Expression of genetic information . . . . . . . . . . . . . . . 28
2.3.1 Transcription . . . . . . . . . . . . . . . . . . . . . . . 30
2.3.2 Translation . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.3 Gene regulation . . . . . . . . . . . . . . . . . . . . . . 35
2.4 The need for high-throughput methods . . . . . . . . . . . . 36
2.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3 Microarrays 39

3.1 Microarrays – tools for gene expression analysis . . . . . . . 39

3.2 Fabrication of microarrays . . . . . . . . . . . . . . . . . . . 41
3.2.1 Deposition . . . . . . . . . . . . . . . . . . . . . . . . 41
3.2.1.1 The Illumina technology . . . . . . . . . . . 42
3.2.2 In situ synthesis . . . . . . . . . . . . . . . . . . . . . 48
3.2.3 A brief comparison of cDNA and oligonucleotide tech-
nologies . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3 Applications of microarrays . . . . . . . . . . . . . . . . . . . 57
3.4 Challenges in using microarrays in gene expression studies . 58

ix
x Contents

3.5 Sources of variability . . . . . . . . . . . . . . . . . . . . . . 63

3.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4 Reliability and reproducibility issues in DNA microarray

measurements 69

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.2 What is expected from microarrays? . . . . . . . . . . . . . . 70
4.3 Basic considerations of microarray measurements . . . . . . 70
4.4 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.5 Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.6 Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.7 Cross-platform consistency . . . . . . . . . . . . . . . . . . . 78
4.8 Sources of inaccuracy and inconsistencies in microarray mea-
surements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.9 The MicroArray Quality Control (MAQC) project . . . . . . 85
4.10 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

5 Image processing 89

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
5.2 Basic elements of digital imaging . . . . . . . . . . . . . . . . 90
5.3 Microarray image processing . . . . . . . . . . . . . . . . . . 95
5.4 Image processing of cDNA microarrays . . . . . . . . . . . . 96
5.4.1 Spot finding . . . . . . . . . . . . . . . . . . . . . . . . 99
5.4.2 Image segmentation . . . . . . . . . . . . . . . . . . . 100
5.4.3 Quantification . . . . . . . . . . . . . . . . . . . . . . 106
5.4.4 Spot quality assessment . . . . . . . . . . . . . . . . . 111
5.5 Image processing of Affymetrix arrays . . . . . . . . . . . . . 113
5.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

6 Introduction to R 119

6.1 Introduction to R . . . . . . . . . . . . . . . . . . . . . . . . 119

6.1.1 About R and Bioconductor . . . . . . . . . . . . . . . 119
6.1.2 Repositories for R and Bioconductor . . . . . . . . . . 120
6.1.3 The working setup for R . . . . . . . . . . . . . . . . . 121
6.1.4 Getting help in R . . . . . . . . . . . . . . . . . . . . . 122
6.2 The basic concepts . . . . . . . . . . . . . . . . . . . . . . . . 122
6.2.1 Elementary computations . . . . . . . . . . . . . . . . 122
6.2.2 Variables and assignments . . . . . . . . . . . . . . . . 125
6.2.3 Expressions and objects . . . . . . . . . . . . . . . . . 126
6.3 Data structures and functions . . . . . . . . . . . . . . . . . 128
6.3.1 Vectors and vector operations . . . . . . . . . . . . . . 128
6.3.2 Referencing vector elements . . . . . . . . . . . . . . . 131
 Contents xi

6.3.3 Functions . . . . . . . . . . . . . . . . . . . . . . . . . 133

6.3.4 Creating vectors . . . . . . . . . . . . . . . . . . . . . 135
6.3.5 Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . 137
6.3.6 Lists . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6.3.7 Data frames . . . . . . . . . . . . . . . . . . . . . . . . 141
6.4 Other capabilities . . . . . . . . . . . . . . . . . . . . . . . . 144
6.4.1 More advanced indexing . . . . . . . . . . . . . . . . . 144
6.4.2 Missing values . . . . . . . . . . . . . . . . . . . . . . 145
6.4.3 Reading and writing files . . . . . . . . . . . . . . . . 148
6.4.4 Conditional selection and indexing . . . . . . . . . . . 150
6.4.5 Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . 151
6.4.6 Implicit loops . . . . . . . . . . . . . . . . . . . . . . . 154
6.5 The R environment . . . . . . . . . . . . . . . . . . . . . . . 159
6.5.1 The search path: attach and detach . . . . . . . . . . 159
6.5.2 The workspace . . . . . . . . . . . . . . . . . . . . . . 161
6.5.3 Packages . . . . . . . . . . . . . . . . . . . . . . . . . 163
6.5.4 Built-in data . . . . . . . . . . . . . . . . . . . . . . . 165
6.6 Installing Bioconductor . . . . . . . . . . . . . . . . . . . . . 165
6.7 Graphics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
6.8 Control structures in R . . . . . . . . . . . . . . . . . . . . . 169
6.8.1 Conditional statements . . . . . . . . . . . . . . . . . 170
6.8.2 Pre-test loops . . . . . . . . . . . . . . . . . . . . . . . 171
6.8.3 Counting loops . . . . . . . . . . . . . . . . . . . . . . 172
6.8.4 Breaking out of loops . . . . . . . . . . . . . . . . . . 173
6.8.5 Post-test loops . . . . . . . . . . . . . . . . . . . . . . 173
6.9 Programming in R versus C/C++/Java . . . . . . . . . . . . 174
6.9.1 R is “forgiving” – which can be bad . . . . . . . . . . . 174
6.9.2 Weird syntax errors . . . . . . . . . . . . . . . . . . . 175
6.9.3 Programming style . . . . . . . . . . . . . . . . . . . . 179
6.10 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
6.11 Solved Exercises . . . . . . . . . . . . . . . . . . . . . . . . . 183
6.12 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

7 Bioconductor: principles and illustrations 193

7.1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

7.2 The portal . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
7.2.1 The main resource categories . . . . . . . . . . . . . . 195
7.2.2 Working with the software repository . . . . . . . . . 195
7.3 Some explorations and analyses . . . . . . . . . . . . . . . . 197
7.3.1 The representation of microarray data . . . . . . . . . 197
7.3.2 The annotation of a microarray platform . . . . . . . 199
7.3.3 Predictive modeling using microarray data . . . . . . 203
7.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
xii Contents

8 Elements of statistics 207

8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

8.2 Some basic concepts . . . . . . . . . . . . . . . . . . . . . . . 208
8.2.1 Populations versus samples . . . . . . . . . . . . . . . 208
8.2.2 Parameters versus statistics . . . . . . . . . . . . . . . 209
8.3 Elementary statistics . . . . . . . . . . . . . . . . . . . . . . 211
8.3.1 Measures of central tendency: mean, mode, and median 211
8.3.1.1 Mean . . . . . . . . . . . . . . . . . . . . . . 211
8.3.1.2 Mode . . . . . . . . . . . . . . . . . . . . . . 212
8.3.1.3 Median, percentiles, and quantiles . . . . . . 213
8.3.1.4 Characteristics of the mean, mode, and me-
dian . . . . . . . . . . . . . . . . . . . . . . . 214
8.3.2 Measures of variability . . . . . . . . . . . . . . . . . . 215
8.3.2.1 Range . . . . . . . . . . . . . . . . . . . . . . 215
8.3.2.2 Variance . . . . . . . . . . . . . . . . . . . . 216
8.3.3 Some interesting data manipulations . . . . . . . . . . 218
8.3.4 Covariance and correlation . . . . . . . . . . . . . . . 219
8.3.5 Interpreting correlations . . . . . . . . . . . . . . . . . 223
8.3.6 Measurements, errors, and residuals . . . . . . . . . . 230
8.4 Degrees of freedom . . . . . . . . . . . . . . . . . . . . . . . 231
8.4.1 Degrees of freedom as independent error estimates . . 232
8.4.2 Degrees of freedom as number of additional measure-
ments . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
8.4.3 Degrees of freedom as observations minus restrictions 233
8.4.4 Degrees of freedom as measurements minus model pa-
rameters . . . . . . . . . . . . . . . . . . . . . . . . . . 234
8.4.5 Degrees of freedom as number of measurements we can
change . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
8.4.6 Data split between estimating variability and model pa-
rameters . . . . . . . . . . . . . . . . . . . . . . . . . . 235
8.4.7 A geometrical perspective . . . . . . . . . . . . . . . . 235
8.4.8 Calculating the number of degrees of freedom . . . . . 236
8.4.8.1 Estimating k quantities from n measurements 236
8.4.9 Calculating the degrees of freedom for an n × m table 237
8.5 Probabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
8.5.1 Computing with probabilities . . . . . . . . . . . . . . 243
8.5.1.1 Addition rule . . . . . . . . . . . . . . . . . . 243
8.5.1.2 Conditional probabilities . . . . . . . . . . . 244
8.5.1.3 General multiplication rule . . . . . . . . . . 247
8.6 Bayes’ theorem . . . . . . . . . . . . . . . . . . . . . . . . . . 247
8.7 Testing for (or predicting) a disease . . . . . . . . . . . . . . 250
8.7.1 Basic criteria: accuracy, sensitivity, specificity, PPV,
NPV . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
 Contents xiii

8.7.2 More about classification criteria: prevalence, incidence,

and various interdependencies . . . . . . . . . . . . . . 253
8.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
8.9 Solved problems . . . . . . . . . . . . . . . . . . . . . . . . . 257
8.10 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

9 Probability distributions 261

9.1 Probability distributions . . . . . . . . . . . . . . . . . . . . 261

9.1.1 Discrete random variables . . . . . . . . . . . . . . . . 262
9.1.2 The discrete uniform distribution . . . . . . . . . . . . 265
9.1.3 Binomial distribution . . . . . . . . . . . . . . . . . . 266
9.1.4 Poisson distribution . . . . . . . . . . . . . . . . . . . 275
9.1.5 The hypergeometric distribution . . . . . . . . . . . . 278
9.1.6 Continuous random variables . . . . . . . . . . . . . . 281
9.1.7 The continuous uniform distribution . . . . . . . . . . 283
9.1.8 The normal distribution . . . . . . . . . . . . . . . . . 283
9.1.9 Using a distribution . . . . . . . . . . . . . . . . . . . 287
9.2 Central limit theorem . . . . . . . . . . . . . . . . . . . . . . 291
9.3 Are replicates useful? . . . . . . . . . . . . . . . . . . . . . . 292
9.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
9.5 Solved problems . . . . . . . . . . . . . . . . . . . . . . . . . 295
9.6 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

10 Basic statistics in R 299

10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 299

10.2 Descriptive statistics in R . . . . . . . . . . . . . . . . . . . . 300
10.2.1 Mean, median, range, variance, and standard deviation 300
10.2.2 Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
10.2.3 More built-in R functions for descriptive statistics . . 305
10.2.4 Covariance and correlation . . . . . . . . . . . . . . . 307
10.3 Probabilities and distributions in R . . . . . . . . . . . . . . 308
10.3.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . 308
10.3.2 Empirical probabilities . . . . . . . . . . . . . . . . . . 309
10.3.3 Standard distributions in R . . . . . . . . . . . . . . . 315
10.3.4 Generating (pseudo-)random numbers . . . . . . . . . 316
10.3.5 Probability density functions . . . . . . . . . . . . . . 316
10.3.6 Cumulative distribution functions . . . . . . . . . . . 317
10.3.7 Quantiles . . . . . . . . . . . . . . . . . . . . . . . . . 319
10.3.7.1 The normal distribution . . . . . . . . . . . . 321
10.3.7.2 The binomial distribution . . . . . . . . . . . 324
10.3.8 Using built-in distributions in R . . . . . . . . . . . . 326
10.4 Central limit theorem . . . . . . . . . . . . . . . . . . . . . . 329
10.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
xiv Contents

10.6 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

11 Statistical hypothesis testing 337

11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

11.2 The framework . . . . . . . . . . . . . . . . . . . . . . . . . . 338
11.3 Hypothesis testing and significance . . . . . . . . . . . . . . 341
11.3.1 One-tailed testing . . . . . . . . . . . . . . . . . . . . 342
11.3.2 Two-tailed testing . . . . . . . . . . . . . . . . . . . . 346
11.4 “I do not believe God does not exist” . . . . . . . . . . . . . 348
11.5 An algorithm for hypothesis testing . . . . . . . . . . . . . . 350
11.6 Errors in hypothesis testing . . . . . . . . . . . . . . . . . . . 351
11.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
11.8 Solved problems . . . . . . . . . . . . . . . . . . . . . . . . . 356

12 Classical approaches to data analysis 359

12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

12.2 Tests involving a single sample . . . . . . . . . . . . . . . . . 360
12.2.1 Tests involving the mean. The t distribution. . . . . . 360
12.2.2 Choosing the number of replicates . . . . . . . . . . . 366
12.2.3 Tests involving the variance (σ 2 ). The chi-square distri-
bution . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
12.2.4 Confidence intervals for standard deviation/variance . 374
12.3 Tests involving two samples . . . . . . . . . . . . . . . . . . . 375
12.3.1 Comparing variances. The F distribution. . . . . . . . 375
12.3.2 Comparing means . . . . . . . . . . . . . . . . . . . . 380
12.3.2.1 Equal variances . . . . . . . . . . . . . . . . 384
12.3.2.2 Unequal variances . . . . . . . . . . . . . . . 386
12.3.2.3 Paired testing . . . . . . . . . . . . . . . . . 387
12.3.3 Confidence intervals for the difference of means µ1 − µ2 388
12.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
12.5 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

13 Analysis of Variance – ANOVA 393

13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

13.1.1 Problem definition and model assumptions . . . . . . 393
13.1.2 The “dot” notation . . . . . . . . . . . . . . . . . . . . 397
13.2 One-way ANOVA . . . . . . . . . . . . . . . . . . . . . . . . 398
13.2.1 One-way Model I ANOVA . . . . . . . . . . . . . . . . 398
13.2.1.1 Partitioning the Sum of Squares . . . . . . . 399
13.2.1.2 Degrees of freedom . . . . . . . . . . . . . . . 401
13.2.1.3 Testing the hypotheses . . . . . . . . . . . . 401
13.2.2 One-way Model II ANOVA . . . . . . . . . . . . . . . 405
 Contents xv

13.3 Two-way ANOVA . . . . . . . . . . . . . . . . . . . . . . . . 408

13.3.1 Randomized complete block design ANOVA . . . . . . 409
13.3.2 Comparison between one-way ANOVA and randomized
block design ANOVA . . . . . . . . . . . . . . . . . . 412
13.3.3 Some examples . . . . . . . . . . . . . . . . . . . . . . 413
13.3.4 Factorial design two-way ANOVA . . . . . . . . . . . 417
13.3.5 Data analysis plan for factorial design ANOVA . . . . 422
13.3.6 Reference formulae for factorial design ANOVA . . . . 423
13.4 Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . 423
13.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
13.6 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

14 Linear models in R 431

14.1 Introduction and model formulation . . . . . . . . . . . . . . 431

14.2 Fitting linear models in R . . . . . . . . . . . . . . . . . . . 433
14.3 Extracting information from a fitted model: testing hypotheses
and making predictions . . . . . . . . . . . . . . . . . . . . . 437
14.4 Some limitations of linear models . . . . . . . . . . . . . . . 438
14.5 Dealing with multiple predictors and interactions in the linear
models, and interpreting model coefficients . . . . . . . . . . 441
14.5.1 Details on the design matrix creation and coefficients
estimation in linear models . . . . . . . . . . . . . . . 443
14.5.2 ANOVA using linear models . . . . . . . . . . . . . . . 445
14.5.2.1 One-way Model I ANOVA . . . . . . . . . . 446
14.5.2.2 Randomized block design ANOVA . . . . . . 451
14.5.3 Practical linear models for analysis of microarray data 452
14.5.4 A two-group comparison gene expression analysis using
a simple t -test . . . . . . . . . . . . . . . . . . . . . . 453
14.5.5 Differential expression using the limma library of Bio-
conductor . . . . . . . . . . . . . . . . . . . . . . . . . 455
14.5.5.1 Two group comparison with single-channel
data . . . . . . . . . . . . . . . . . . . . . . . 455
14.5.5.2 Multiple contrasts with single-channel data . 457
14.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

15 Experiment design 461

15.1 The concept of experiment design . . . . . . . . . . . . . . . 462

15.2 Comparing varieties . . . . . . . . . . . . . . . . . . . . . . . 462
15.3 Improving the production process . . . . . . . . . . . . . . . 464
15.4 Principles of experimental design . . . . . . . . . . . . . . . . 466
15.4.1 Replication . . . . . . . . . . . . . . . . . . . . . . . . 466
15.4.2 Randomization . . . . . . . . . . . . . . . . . . . . . . 469
15.4.3 Blocking . . . . . . . . . . . . . . . . . . . . . . . . . . 470
xvi Contents

15.5 Guidelines for experimental design . . . . . . . . . . . . . . . 470

15.6 A short synthesis of statistical experiment designs . . . . . . 472
15.6.1 The fixed effect design . . . . . . . . . . . . . . . . . . 473
15.6.2 Randomized block design . . . . . . . . . . . . . . . . 474
15.6.3 Balanced incomplete block design . . . . . . . . . . . . 474
15.6.4 Latin square design . . . . . . . . . . . . . . . . . . . 475
15.6.5 Factorial design . . . . . . . . . . . . . . . . . . . . . . 475
15.6.6 Confounding in the factorial design . . . . . . . . . . . 478
15.7 Some microarray specific experiment designs . . . . . . . . . 479
15.7.1 The Jackson Lab approach . . . . . . . . . . . . . . . 479
15.7.2 Ratios and flip-dye experiments . . . . . . . . . . . . . 482
15.7.3 Reference design versus loop design . . . . . . . . . . . 484
15.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

16 Multiple comparisons 489

16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

16.2 The problem of multiple comparisons . . . . . . . . . . . . . 490
16.3 A more precise argument . . . . . . . . . . . . . . . . . . . . 497
16.4 Corrections for multiple comparisons . . . . . . . . . . . . . 499
16.4.1 The Šidák correction . . . . . . . . . . . . . . . . . . . 499
16.4.2 The Bonferroni correction . . . . . . . . . . . . . . . . 500
16.4.3 Holm’s step-wise correction . . . . . . . . . . . . . . . 501
16.4.4 The false discovery rate (FDR) . . . . . . . . . . . . . 502
16.4.5 Permutation correction . . . . . . . . . . . . . . . . . 503
16.4.6 Significance analysis of microarrays (SAM) . . . . . . 505
16.4.7 On permutation-based methods . . . . . . . . . . . . . 506
16.5 Corrections for multiple comparisons in R . . . . . . . . . . . 506
16.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

17 Analysis and visualization tools 513

17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

17.2 Box plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
17.3 Gene pies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
17.4 Scatter plots . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
17.4.1 Scatter plots in R . . . . . . . . . . . . . . . . . . . . 523
17.4.2 Scatter plot limitations . . . . . . . . . . . . . . . . . 524
17.4.3 Scatter plot summary . . . . . . . . . . . . . . . . . . 527
17.5 Volcano plots . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
17.5.1 Volcano plots in R . . . . . . . . . . . . . . . . . . . . 528
17.6 Histograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
17.6.1 Histograms summary . . . . . . . . . . . . . . . . . . . 540
17.7 Time series . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540
17.8 Time series plots in R . . . . . . . . . . . . . . . . . . . . . . 541
 Contents xvii

17.9 Principal component analysis (PCA) . . . . . . . . . . . . . 548

17.9.1 PCA limitations . . . . . . . . . . . . . . . . . . . . . 556
17.9.2 Principal component analysis in R . . . . . . . . . . . 557
17.9.3 PCA summary . . . . . . . . . . . . . . . . . . . . . . 558
17.10 Independent component analysis (ICA) . . . . . . . . . . . . 561
17.11 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

18 Cluster analysis 565

18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

18.2 Distance metric . . . . . . . . . . . . . . . . . . . . . . . . . 566
18.2.1 Euclidean distance . . . . . . . . . . . . . . . . . . . . 567
18.2.2 Manhattan distance . . . . . . . . . . . . . . . . . . . 568
18.2.3 Chebychev distance . . . . . . . . . . . . . . . . . . . 570
18.2.4 Angle between vectors . . . . . . . . . . . . . . . . . . 571
18.2.5 Correlation distance . . . . . . . . . . . . . . . . . . . 571
18.2.6 Squared Euclidean distance . . . . . . . . . . . . . . . 572
18.2.7 Standardized Euclidean distance . . . . . . . . . . . . 573
18.2.8 Mahalanobis distance . . . . . . . . . . . . . . . . . . 575
18.2.9 Minkowski distance . . . . . . . . . . . . . . . . . . . . 575
18.2.10 When to use what distance . . . . . . . . . . . . . . . 576
18.2.11 A comparison of various distances . . . . . . . . . . . 578
18.3 Clustering algorithms . . . . . . . . . . . . . . . . . . . . . . 579
18.3.1 k -means clustering . . . . . . . . . . . . . . . . . . . . 582
18.3.1.1 Characteristics of the k-means clustering . . 584
18.3.1.2 Cluster quality assessment . . . . . . . . . . 586
18.3.1.3 Number of clusters in k-means . . . . . . . . 591
18.3.1.4 Algorithm complexity . . . . . . . . . . . . . 591
18.3.2 Hierarchical clustering . . . . . . . . . . . . . . . . . . 592
18.3.2.1 Inter-cluster distances and algorithm complex-
ity . . . . . . . . . . . . . . . . . . . . . . . . 594
18.3.2.2 Top-down versus bottom-up . . . . . . . . . 595
18.3.2.3 Cutting tree diagrams . . . . . . . . . . . . . 597
18.3.2.4 An illustrative example . . . . . . . . . . . . 599
18.3.2.5 Hierarchical clustering summary . . . . . . . 601
18.3.3 Kohonen maps or self-organizing feature maps (SOFM) 603
18.4 Partitioning around medoids (PAM) . . . . . . . . . . . . . . 612
18.5 Biclustering . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
18.5.1 Types of biclusters . . . . . . . . . . . . . . . . . . . . 615
18.5.2 Biclustering algorithms . . . . . . . . . . . . . . . . . 616
18.5.3 Differential biclustering . . . . . . . . . . . . . . . . . 618
18.5.4 Biclustering summary . . . . . . . . . . . . . . . . . . 619
18.6 Clustering in R . . . . . . . . . . . . . . . . . . . . . . . . . . 619
18.6.1 Partition around medoids (PAM) in R . . . . . . . . . 627
18.6.2 Biclustering in R . . . . . . . . . . . . . . . . . . . . . 629
xviii Contents

18.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630

19 Quality control 633

19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

19.2 Quality control for Affymetrix data . . . . . . . . . . . . . . 634
19.2.1 Reading raw data (.CEL files) . . . . . . . . . . . . . . 634
19.2.2 Intensity distributions . . . . . . . . . . . . . . . . . . 635
19.2.3 Box plots . . . . . . . . . . . . . . . . . . . . . . . . . 637
19.2.4 Probe intensity images . . . . . . . . . . . . . . . . . . 637
19.2.5 Quality control metrics . . . . . . . . . . . . . . . . . 639
19.2.6 RNA degradation curves . . . . . . . . . . . . . . . . . 645
19.2.7 Quality control plots . . . . . . . . . . . . . . . . . . . 647
19.2.8 Probe-level model (PLM) fitting. RLE and NUSE plots 652
19.3 Quality control of Illumina data . . . . . . . . . . . . . . . . 658
19.3.1 Reading Illumina data . . . . . . . . . . . . . . . . . . 658
19.3.2 Bead-summary data . . . . . . . . . . . . . . . . . . . 661
19.3.2.1 Raw probe data import, visualization, and
quality assessment using “beadarray” . . . . 661
19.3.2.2 Raw probe data import, visualization, and
quality assessment using “lumi” . . . . . . . . 663
19.3.3 Bead-level data . . . . . . . . . . . . . . . . . . . . . . 667
19.3.3.1 Raw bead data import and assessment . . . 667
19.3.3.2 Summarizing from bead-level to probe-level
data . . . . . . . . . . . . . . . . . . . . . . . 688
19.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689

20 Data preprocessing and normalization 691

20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 691

20.2 General preprocessing techniques . . . . . . . . . . . . . . . . 692
20.2.1 The log transform . . . . . . . . . . . . . . . . . . . . 692
20.2.2 Combining replicates and eliminating outliers . . . . . 694
20.2.3 Array normalization . . . . . . . . . . . . . . . . . . . 696
20.2.3.1 Dividing by the array mean . . . . . . . . . . 699
20.2.3.2 Subtracting the mean . . . . . . . . . . . . . 699
20.2.3.3 Using control spots/genes . . . . . . . . . . . 701
20.2.3.4 Iterative linear regression . . . . . . . . . . . 701
20.2.3.5 Other aspects of array normalization . . . . 702
20.3 Normalization issues specific to cDNA data . . . . . . . . . . 702
20.3.1 Background correction . . . . . . . . . . . . . . . . . . 702
20.3.1.1 Local background correction . . . . . . . . . 702
20.3.1.2 Sub-grid background correction . . . . . . . 703
20.3.1.3 Group background correction . . . . . . . . . 703
20.3.1.4 Background correction using blank spots . . 703
 Contents xix

20.3.1.5 Background correction using control spots . 703

20.3.2 Other spot level preprocessing . . . . . . . . . . . . . 704
20.3.3 Color normalization . . . . . . . . . . . . . . . . . . . 704
20.3.3.1 Curve fitting and correction . . . . . . . . . 706
20.3.3.2 LOWESS/LOESS normalization . . . . . . . 708
20.3.3.3 Piece-wise normalization . . . . . . . . . . . 711
20.3.3.4 Other approaches to cDNA data normaliza-
tion . . . . . . . . . . . . . . . . . . . . . . . 713
20.4 Normalization issues specific to Affymetrix data . . . . . . . 713
20.4.1 Background correction . . . . . . . . . . . . . . . . . . 713
20.4.2 Signal calculation . . . . . . . . . . . . . . . . . . . . . 716
20.4.2.1 Ideal mismatch . . . . . . . . . . . . . . . . . 716
20.4.2.2 Probe values . . . . . . . . . . . . . . . . . . 717
20.4.2.3 Scaled probe values . . . . . . . . . . . . . . 718
20.4.3 Detection calls . . . . . . . . . . . . . . . . . . . . . . 719
20.4.4 Relative expression values . . . . . . . . . . . . . . . . 720
20.5 Other approaches to the normalization of Affymetrix data . 720
20.5.1 Cyclic Loess . . . . . . . . . . . . . . . . . . . . . . . . 720
20.5.2 The model-based dChip approach . . . . . . . . . . . 721
20.5.3 The Robust Multi-Array Analysis (RMA) . . . . . . . 722
20.5.4 Quantile normalization . . . . . . . . . . . . . . . . . . 722
20.6 Useful preprocessing and normalization sequences . . . . . . 725
20.7 Normalization procedures in R . . . . . . . . . . . . . . . . . 726
20.7.1 Normalization functions and procedures for Affymetrix
data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 726
20.7.2 Background adjustment and various types of normaliza-
tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732
20.7.3 Summarization . . . . . . . . . . . . . . . . . . . . . . 733
20.8 Batch preprocessing . . . . . . . . . . . . . . . . . . . . . . . 736
20.9 Normalization functions and procedures for Illumina data . . 737
20.10Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741
20.11Appendix: A short primer on logarithms . . . . . . . . . . . 744

21 Methods for selecting differentially expressed genes 747

21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

21.2 Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749
21.3 Fold change . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
21.3.1 Description . . . . . . . . . . . . . . . . . . . . . . . . 751
21.3.2 Characteristics . . . . . . . . . . . . . . . . . . . . . . 752
21.4 Unusual ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
21.4.1 Description . . . . . . . . . . . . . . . . . . . . . . . . 754
21.4.2 Characteristics . . . . . . . . . . . . . . . . . . . . . . 755
21.5 Hypothesis testing, corrections for multiple comparisons, and
resampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756
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