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Foreword, xiii Preface, xv Acknowledgements, xix About the Companion Website, xxi 1 Introduction to R statistical environment, 1 Why R?, 1 Installing R, 2 Interacting with R, 2 Graphical interfaces and integrated development environment (IDE) integration, 3 Scripting and sourcing, 3 The R history and the R environment file, 4 Packages and package repositories, 4 Comprehensive R Archive Network, 5 Bioconductor, 6 Working with data, 7 Basic operations in R, 8 Some basics of graphics in R, 10 Getting help in R, 12 Files for practicing, 13 Study exercises and questions, 14 References, 14 Webliography, 15 2 Simple sequence analysis, 17 Sequence files, 17 FASTA sequence format, 18 GenBank flat file format, 19 Reading sequence files into R, 20 Obtaining sequences from remote databases, 21 Seqinr package, 21 Ape package, 22 Descriptive statistics of nucleotide sequences, 24 Descriptive statistics of proteins, 28 Aligned sequences, 31 Visualization of genes and transcripts in a professional way, 34 Files for practicing, 37 Study exercises and questions, 38 References, 38 Webliography, 39 Packages, 40 3 Annotating gene groups, 41 Enrichment analysis: an overview, 41 Overview of two different methods, 41 Enrichment analysis results, 42 Common aspects of the two different approaches, 43 Overrepresentation analysis, 46 Hypergeometric test using GOstats, 47 ORA analysis using topGO, 48 Enrichment analysis of microarray sets with topGO, 51 Gene set enrichment analysis, 52 GSEA with R, 56 Files for practicing, 61 Study exercises and questions, 61 References, 62 Webliography, 62 Packages, 63 4 Next-generation sequencing: introduction and genomic applications, 65 High-throughput sequencing background, 65 Experimental background, 66 Single-end and paired-end sequencing reads, 67 Assemble reads, 69 How many reads? Depth of coverage, 71 Storing data in files, 72 FASTQ, 72 SAM and BAM files, 76 Variant call format files, 77 General data analysis workflow, 77 Data processing considerations, 78 Quality checking and screening read sequences, 80 Quality checking for one file, 82 Quality inspection for multiple files in a project, 82 Quality filtering of FASTQ files, 83 Handling alignment files and genomic variants, 84 Alignment and variation visualization, 88 Simple handling of VCF files, 89 Genomic applications: low- and medium-depth sequencing, 91 Aneuploidity sequencing and copy number variation identification, 92 SNP identification and validation, 92 Exome sequencing, 93 Genomic region resequencing, 93 Full genome and metagenome sequencing, 94 Files for practicing, 94 Study exercises and questions, 94 References, 95 Webliography, 97 Packages, 97 5 Quantitative transcriptomics: qRT-PCR, 99 Transcriptome, 99 Polymerase chain reaction, 100 Standards for qPCR, 102 R packages, 104 Understanding delta Ct, 104 Calculation of delta Ct, 105 Requirements for real delta Ct calculations, 107 Absolute quantification, 110 Value prediction, the professional way, 114 Relative quantification using the ddCt method, 115 Comparison of two conditions, 116 Comparison of multiple experimental conditions, 118 Quality control with melting curve, 121 Files for practicing, 123 Study exercises and questions, 123 References, 123 Webliography, 124 Packages, 124 6 Advanced transcriptomics: gene expression microarrays, 125 Microarray analysis: probes and samples, 125 Experimental background, 126 Archiving and publishing microarray data, 128 Minimum information standard, 128 Data preprocessing, 128 Accessing data from CEL files, 129 Quality control, 131 Normalization, 132 Differential gene expression, 133 Annotating results, 136 Creating normalized expression set from Illumina data, 138 Automated data access from GEO, 140 Files for practicing, 142 Study exercises and questions, 142 References, 143 Webliography, 144 Packages, 144 7 Next-generation sequencing in transcriptomics: RNA-seq experiments, 145 High-throughput RNA sequencing background, 145 Experimental background, 145 RNA-seq applications, 146 Differential expression with different resolutions, 147 Preparing count tables, 148 Alignment files to read counts, 148 Differential expression in simple comparison, 151 A naive t-test approach, 151 Single factor analysis with edgeR, 153 Differential expression with DESeq, 156 Complex experimental arrangements, 159 Experimental factors and design matrix, 160 GLM with edgeR, 161 GLMs with DESeq, 162 Heatmap visualization, 163 Files for practicing, 164 Study exercises and questions, 164 References, 165 Webliography, 166 Packages, 166 8 Deciphering the regulome: from ChIP to ChIP-seq, 167 Chromatin immunoprecipitation, 167 Experimental background, 168 Fragment analysis, 168 ChIP data in ENCODE, 169 ChIP with tiling microarrays, 169 High-throughput sequencing of ChIP fragments, 176 Connecting annotation to peaks, 181 Analysis of binding site motifs, 182 Files for practicing, 186 Study exercises and questions, 187 References, 187 Webliography, 188 Packages, 189 9 Inferring regulatory and other networks from gene expression data, 191 Gene regulatory networks, 191 Data for gene network inference, 192 Reconstruction of co-expression networks, 193 Gene regulatory network inference focusing of master regulators, 201 Integrated interpretation of genes with GeneAnswers, 207 Files for practicing, 211 Study exercises and questions, 212 References, 213 Packages, 214 10 Analysis of biological networks, 215 A gentle introduction to networks, 215 Networks and their components and features, 215 Random networks, 220 Biological networks, 221 Files for storing network information, 223 Important network metrics in biology, 227 Distance-based measures, 228 Degree and related measures, 230 Vulnerability, 231 Community structure of a network, 234 Graph visualization, 236 Cytoscape, 240 Files for practicing, 241 Study exercises and questions, 241 References, 242 Webliography, 243 Packages, 243 11 Proteomics: mass spectrometry, 245 Mass spectrometry and proteomics: why and how?, 245 File formats for MS data, 246 Accessing the raw data of published studies, 247 Identification of peptides in the samples, 249 Peptide mass fingerprinting, 249 Peptide identification by using MS/MS spectra, 250 Quantitative proteomics, 254 Getting protein-specific annotation, 258 Files for practicing, 259 Study exercises and questions, 259 References, 259 Webliography, 260 Packages, 260 12 Measuring protein abundance with ELISA, 261 Enzyme-linked immunosorbent assays, 261 Accessing ELISA data, 264 Concentration calculation with a standard curve, 264 Preparing reference data, 267 Fitting linear model, 268 Fitting of a logistic model, 269 Concentration calculations by employing models, 270 Comparative calculations using concentrations, 271 Files for practicing, 277 Study exercises and questions, 277 References, 277 Packages, 278 13 Flow cytometry: counting and sorting stained cells, 279 Theoretical aspects of flow cytometry, 279 Experiment types: diagnosis versus discovery, 280 Measurement arrangements, 281 Fluorescent dyes, 281 Tubes versus plates, 285 Instruments, 285 What about data?, 287 Files, 287 Workflows, 288 Data preprocessing, 289 Handling all samples together, 290 Compensation, 292 Quality assurance, 292 Using workflow objects and transformation, 296 Normalization, 298 Cell population identification, 299 Manual gating, 300 Automatic gating, 304 Relating cell populations to external variables, 305 Reporting results, 307 MIFlowCyt, 307 FlowRepository.org, 308 Files for practicing, 308 Study exercises and questions, 309 References, 309 Webliography, 310 Packages, 310 Glossary, 311 Index, 323