List of Contributors XVII

Part One Sanger DNA Sequencing 1

1 Sanger DNA Sequencing 3
Artem E. Men, Peter Wilson, Kirby Siemering, and Susan Forrest

1.1 The Basics of Sanger Sequencing 3

1.2 Into the Human Genome Project (HGP) and Beyond 6

1.3 Limitations and Future Opportunities 7

1.4 Bioinformatics Holds the Key 8

1.5 Where to Next? 9

Part Two Next-Generation Sequencing: Toward Personalized Medicine 13

2 Illumina Genome Analyzer II System 15
Abizar Lakdawalla and Harper VanSteenhouse

2.1 Library Preparation 15

2.2 Cluster Creation 17

2.3 Sequencing 19

2.4 Paired End Reads 19

2.5 Data Analysis 20

2.6 Applications 21

2.7 Conclusions 26

3 Applied Biosystems SOLiDTM System: Ligation-Based Sequencing 29
Vicki Pandey, Robert C. Nutter, and Ellen Prediger

3.1 Introduction 29

3.2 Overview of the SOLiDTM System 29

3.3 SOLiDTM System Applications 35

3.4 Conclusions 40

4 The Next-Generation Genome Sequencing: 454/Roche GS FLX 43
Lei Du and Michael Egholm

4.1 Introduction 43

4.2 Technology Overview 44

4.3 Software and Bioinformatics 47

4.4 Research Applications 49

5 Polony Sequencing: History, Technology, and Applications 57
Jeremy S. Edwards

5.1 Introduction 57

5.2 History of Polony Sequencing 57

5.3 Polony Sequencing 62

5.4 Applications 69

5.5 Conclusions 75

Part Three The Bottleneck: Sequence Data Analysis 77

6 Next-Generation Sequence Data Analysis 79
Leonard N. Bloksberg

6.1 Why Next-Generation Sequence Analysis is Different? 79

6.2 Strategies for Sequence Searching 80

6.3 What is a "Hit,"and Why it Matters for NGS? 82

6.4 Scoring: Why it is Different for NGS? 83

6.5 Strategies for NGS Sequence Analysis 84

6.6 Subsequent Data Analysis 86

7 DNASTAR.s Next-Generation Software 89
Tim Durfee and Thomas E. Schwei

7.1 Personalized Genomics and Personalized Medicine 89

7.2 Next-Generation DNA Sequencing as the Means to Personalized Genomics 89

7.3 Strengths of Various Platforms 90

7.4 The Computational Challenge 90

7.5 DNASTAR.s Next-Generation Software Solution 91

7.6 Conclusions 94

Part Four Emerging Sequencing Technologies 95

8 Real-Time DNA Sequencing 97
Susan H. Hardin

8.1 Whole Genome Analysis 97

8.2 Personalized Medicine and Pharmacogenomics 97

8.3 Biodefense, Forensics, DNA Testing, and Basic Research 98

8.4 Simple and Elegant: Real-Time DNA Sequencing 98

9 Direct Sequencing by TEM of Z-Substituted DNA Molecules 103
William K. Thomas and William Glover

9.1 Introduction 103

9.2 Logic of Approach 104

9.3 Identification of Optimal Modified Nucleotides for TEM Visual Resolution of DNA Sequences Independent of Polymerization 106

9.4 TEM Substrates and Visualization 107

9.5 Incorporation of Z-Tagged Nucleotides by Polymerases 108

9.6 Current and New Sequencing Technology 109

9.7 Accuracy 111

9.8 Advantages of ZSG.s Proposed DNA Sequencing Technology 111

9.9 Advantages of Significantly Longer Read Lengths 112

10 A Single DNA Molecule Barcoding Method with Applications in DNA Mapping and Molecular Haplotyping 117
Ming Xiao and Pui-Yan Kwok

10.1 Introduction 117

10.2 Critical Techniques in the Single DNA Molecule Barcoding Method 118

10.3 Single DNA Molecule Mapping 120

10.4 Molecular Haplotyping 124

10.5 Discussion 129

11 Optical Sequencing: Acquisition from Mapped Single-Molecule Templates 133
Shiguo Zhou, Louise Pape, and David C. Schwartz

11.1 Introduction 133

11.2 The Optical Sequencing Cycle 135

11.3 Future of Optical Sequencing 148

12 Microchip-Based Sanger Sequencing of DNA 153
Ryan E. Forster, Christopher P. Fredlake, and Annelise E. Barron

12.1 Integrated Microfluidic Devices for Genomic Analysis 154

12.2 Improved Polymer Networks for Sanger Sequencing on Microfluidic Devices 156

12.3 Conclusions 160

Part Five Next-Generation Sequencing: Truly Integrated Genome Analysis 165

13 Multiplex Sequencing of Paired End Ditags for Transcriptome and Genome Analysis 167
Chia-Lin Wei and Yijun Ruan

13.1 Introduction 167

13.2 The Development of Paired End Ditag Analysis 168

13.3 GIS-PET for Transcriptome Analysis 170

13.4 ChIP-PET for Whole Genome Mapping of Transcription Factor Binding Sites and Epigenetic Modifications 173

13.5 ChIA-PET for Whole Genome Identification of Long-Range Interactions 175

13.6 Perspective 179

14 Paleogenomics Using the 454 Sequencing Platform 183
M.Thomas P. Gilbert

14.1 Introduction 183

14.2 The DNA Degradation Challenge 184

14.3 The Effects of DNA Degradation on Paleogenomics 185

14.4 Degradation and Sequencing Accuracy 185

14.5 Sample Contamination 189

14.6 Solutions to DNA Damage 191

14.7 Solutions to Contamination 192

14.8 What Groundwork Remains, and What Does the Future Hold? 195

15 ChIP-seq: Mapping of Protein–DNA Interactions 201
Anthony Peter Fejes and Steven J.M. Jones

15.1 Introduction 201

15.2 History 202

15.3 ChIP-seq Method 202

15.4 Sanger Dideoxy-Based Tag Sequencing 203

15.5 Hybridization-Based Tag Sequencing 205

15.6 Application of Sequencing by Synthesis 206

15.7 Medical Applications of ChIP-seq 209

15.8 Challenges 209

15.9 Future Uses of ChIP-seq 211

16 MicroRNA Discovery and Expression Profiling using Next-Generation Sequencing 217
Eugene Berezikov and Edwin Cuppen

16.1 Background on miRNAs 217

16.2 miRNA Identification 218

16.3 Experimental Approach 219

16.4 Validation 225

16.5 Outlook 226

17 DeepSAGE: Tag-Based Transcriptome Analysis Beyond Microarrays 229
Kåre L. Nielsen, Annabeth H. Petersen, and Jeppe Emmersen

17.1 Introduction 229

17.2 DeepSAGE 231

17.3 Data Analysis 235

17.4 Comparing Tag-Based Transcriptome Profiles 235

17.5 Future Perspectives 238

18 The New Genomics and Personal Genome Information: Ethical Issues 245
Jeantine E. Lunshof

18.1 The New Genomics and Personal Genome Information: Ethical Issues 245

18.2 The New Genomics: What Makes it Special? 245

18.3 Innovation in Ethics: Why do We Need it? 246

18.4 A Proviso: Global Genomics and Local Ethics 247

18.5 Medical Ethics and Hippocratic Confidentiality 247

18.6 Principles of Biomedical Ethics 248

18.7 Clinical Research and Informed Consent 248

18.8 Large-Scale Research Ethics: New Concepts 249

18.9 Personal Genomes 250

18.10 The Personal Genome Project: Consenting to Disclosure 251

References 252

Index 255