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Statistics and Data Analysis for Microarrays using
MATLAB 2nd edition Draghici Digital Instant Download
Author(s): Draghici, Sorin
ISBN(s): 9781439809761, 1439809763
Edition: 2nd ed
File Details: PDF, 130.85 MB
Year: 2011
Language: english
Statistics and Data Analysis
for Microarrays
Using R and Bioconductor
Second Edition
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Algorithms in Bioinformatics: A Practical Exactly Solvable Models of Biological
Introduction Invasion
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Bioinformatics: A Practical Approach Gene Expression Studies Using
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Statistics and Data Analysis
for Microarrays
Using R and Bioconductor
Second Edition

Sorin Drăghici
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
utn|512748|1435358748

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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