Front Cover 1
Clinical Genomics 4
Copyright Page 5
Dedication 6
Contents 8
List of Contributors 12
Foreword 14
Preface 16
Acknowledgments 18
I. Methods 20
1 Overview of Technical Aspects and Chemistries of Next-Generation Sequencing 22
Clinical Molecular Testing: Finer and Finer Resolution 22
Sanger Sequencing 23
Chemistry of Sanger Sequencing, Electrophoresis, Detection 23
Applications in Clinical Genomics 24
Technical Constraints 25
Read Length and Input Requirements 25
Pooled Input DNA Puts a Limit on Sensitivity 26
Cyclic Array Sequencing 26
Illumina Sequencing 27
Library Prep and Sequencing Chemistry 28
Choice of Platforms 29
Phasing 29
SOLiD Sequencing 30
Ion Torrent Sequencing 33
AmpliSeq Library Preparation 35
Roche 454 Genome Sequencers 35
Third-Generation Sequencing Platforms 37
References 37
2 Clinical Genome Sequencing 40
Introduction 40
Next-Generation Sequencing 40
Sequencing in the Clinical Laboratory 41
Applications and Test Information 43
Challenges of Defining a Test Offering That Is Specific to Each Case 44
Laboratory Process, Data Generation, and Quality Control 45
Preanalytical and Quality Laboratory Processes 45
Analytical 46
Bioinformatics 48
Validation 49
Proficiency Testing 50
Interpretation and Reporting 50
Conclusion 53
References 53
3 Targeted Hybrid Capture Methods 56
Introduction 57
Basic Principles of Hybrid Capture-Based NGS 57
Specimen Requirements and DNA Preparation 57
Determining the Target ROI 58
Designing Capture Baits 58
General Overview of Library Preparation 58
Coverage and Uniformity 60
Specificity and Sensitivity 60
Obstacles of Target Capture 60
Library Complexity 61
Hybrid Capture-Based Target Enrichment Strategies 62
Solid-Phase Hybrid Capture 62
Solution-Based Hybrid Capture 63
Molecular Inversion Probes 65
Comparison of Targeted Hybrid Capture Enrichment Strategies 66
Amplification-Based Enrichment Versus Capture-Based Enrichment 66
Clinical Applications of Target Capture Enrichment 67
Exome Capture 67
Selected Gene Panels 69
Disease-Associated Exome Testing 70
Variant Detection 70
Practical and Operational Considerations 71
Workflow and TAT 71
Conclusions 72
References 72
4 Amplification-Based Methods 76
Introduction 76
Principles of Amplification-Based Targeted NGS 77
Sequencing Workflow 77
Samples Requirements 78
Nucleic Acids Preparation 78
Primer Design for Multiplex PCR 79
Library Preparation and Amplification 80
Other Amplification-Based Target Enrichment Approaches 81
Comparison of Amplification- and Capture-Based Methods 81
Clinical Applications 83
Conclusion 85
References 85
5 Emerging DNA Sequencing Technologies 88
Introduction 89
Third-Generation Sequencing Approaches 90
Single-Molecule Real-Time (SMRT) DNA Sequencing 90
Heliscope Genetic Analysis System 91
Fourth-Generation Sequencing 92
Nanopore Sequencing 92
Selected Novel Technologies 93
In Situ DNA Sequencing 93
Transmission Electron Microscopy 93
Electronic Sequencing 94
Summary 94
References 94
6 RNA-Sequencing and Methylome Analysis 96
Introduction 97
Approaches to Analysis of RNA 97
Microarray Analysis of Differential Gene Expression 97
Next-Generation Methods of RNA-Seq 97
Workflow 98
Typical RNA-Seq Protocol 98
Bioinformatic Analyses of Sequence Generated from RNA-Seq Experiment 98
Initial Processing of Raw Reads: Quality Assessment 98
Read Mapping Strategies 100
De Novo Read Assembly 100
Read Alignment 100
Alignment to Reference Genome 101
RNA-Seq Variant Calling and Filtering 101
Expression Estimation: Summarization, Normalization, and Differential Expression 102
Differential Expression 102
Fusion Detection 102
Depth of Coverage Issues 103
Utility of RNA-Seq to Characterize Alternative Splicing Events 103
Utility of RNA-Seq for Genomic Structural Variant Detection 103
RNA-Seq: Challenges, Pitfalls, and Opportunities in Clinical Applications 104
Methylome Sequencing 104
Conclusions 105
References 105
List of Acronyms and Abbreviations 107
II. Bioinformatics 108
7 Base Calling, Read Mapping, and Coverage Analysis 110
Introduction 111
Library Preparation and Amplification 111
Base Calling 111
Read Mapping 112
Platform-Specific Base Calling Methods 113
Illumina: Platform and Run Metrics 114
Density 114
Intensity by Cycle 115
QScore Distribution 115
QScore Heatmap 115
IVC Plot 115
%Phasing/Prephasing 115
PhiX-Based Quality Metrics 115
Torrent: Platform and Run Metrics 116
Loading or ISP Density 116
Live ISPs 116
Library and Test ISPs 116
Key Signal 116
Clonal 116
Usable Sequence 116
Test Fragment Metrics 117
Illumina: Base Calling 117
Template Generation 117
Base Calling 117
Quality Scoring 118
Torrent: Base Calling 118
Key Processes 118
Postprocessing 119
Intrinsic and Platform-Specific Sources of Error 119
Read Mapping 119
Reference Genome 119
NGS Alignment Tools 120
MAQ 120
Bowtie 120
BWA 120
Novoalign 120
MOSAIK 121
Isaac 121
TMAP 121
Sequence Read and Alignment Formats 121
Sequence Alignment Factors 121
Alignment Processing 122
Coverage Analysis: Metrics for Assessing Genotype Quality 122
Performance and Diagnostic Metrics 122
Total Read Number 123
Percent of Mapped Reads 123
Percent of Read Pairs Aligned 123
Percent of Reads in the Exome or Target Region 123
Library Fragment Length 123
Depth of Coverage 124
Percent of Unique Reads 124
Target Coverage Grap 125
Summary 125
References 126
8 Single Nucleotide Variant Detection Using Next Generation Sequencing 128
Introduction 129
Sources of SNVs 130
Endogenous Sources of Damage Leading to SNVs 130
Reactive Oxygen Species 130
Spontaneous Chemical Reactions 130
Metal Ions 130
Errors in DNA Replication 131
Exogenous Sources of Damage Leading to SNVs 131
Chemical Mutagens 131
Radiation 131
Consequences of SNVs 132
SNVs in Coding Regions 132
Synonymous SNVs 132
Missense SNVs 132
Nonsense SNVs 132
Consequences of SNVs on RNA Processing 132
Altered RNA Splicing 132
SNVs in Regulatory Regions 133
Technical Issues 133
Platform 133
Target Size 133
Target Enrichment Approach 134
Library Complexity 135
Depth of Sequencing 135
Anticipated Sample Purity 135
Sample Type 136
Bioinformatic Approaches for SNV Calling 136
Parameters Used for SNV Detection 137
High Sensitivity Tools 140
Tumor/Normal Analyses 140
Implications for Clinical NGS 140
Orthogonal Validation 141
Interpretation of SNVs 141
Online Resources and Databases 141
Prediction Tools for Missense Variants 141
Prediction Tools for Possible Splice Effects 141
Kindred Testing 142
Paired Tumor-Normal Testing 142
Reporting 142
Summary 142
References 143
9 Insertions and Deletions (Indels) 148
Overview of Insertion/Deletion Events (Indels) 149
Introduction 149
Indel Definition and Relationship to Other Classes of Mutations 149
Testing for Indels in Constitutional and Somatic Disease 150
Sources, Frequency, and Consequences of Indels 152
Mechanisms of Indel Generation 152
Slipped Strand Mispairing (Polymerase Slippage) 152
Secondary Structure Formation 153
Imperfect Double-Strand DNA Break Repair 153
Defective Mismatch Repair 153
Unequal Meiotic Recombination 153
Frequency of Indels in Human Genomes 153
Functional Consequences 155
Decreased Transcription 155
Abnormal Protein Aggregation 156
Microsatellite Instability/Rapid Repeat Expansion 156
Altered Splicing 156
Frameshift 156
In-Frame, Decreased Protein Activity 157
In-Frame, Increased Protein Activity 157
Synonymous, Missense, and Nonsense 157
Predicting Functional Effects of Novel Indels 157
Technical Issues That Impact Indel Detection by NGS 158
Sequencing Platform Chemistry 158
Sequence Read Type and Alignment 159
Library Preparation Technique 160
Depth of Coverage 160
Assay Design 160
Specimen Issues That Impact Indel Detection by NGS 160
Specimen Cellularity and Heterogeneity 161
Library Complexity 161
Bioinformatics Approaches to NGS Indel Detection 161
General Bioinformatics Approaches to Indel Detection and Annotation 161
Local Realignment 161
Left Alignment 162
Probabilistic Modeling Using Mapped Reads 163
Split-Read Analysis 164
Sensitivity and Specificity Issues 165
Indel Length 165
Indel Annotation 165
Definition of Indel “Truth” 166
Reference Standards 166
Summary 167
References 167
10 Translocation Detection Using Next-Generation Sequencing 170
Introduction to Translocations 171
Discovery of Translocations in Human Disease 171
Mechanisms of Translocation Formation 171
Translocations in Human Disease 172
Translocations in Hematologic Malignancies 172
Translocations in Leukemias 172
Translocations in Lymphomas 173
Translocations in Solid Tumors 173
Sarcomas 173
Carcinomas 174
Translocations in Inherited Disorders 174
Developmental Delay 174
Recurrent Miscarriages 174
Hereditary Cancer Syndromes 174
Translocation Detection 175
Conventional Methods 175
Translocation Detection by Whole Genome DNA Sequencing 175
Translocation Detection by Targeted DNA Sequencing 175
Translocation Detection by RNA-Seq 177
Informatic Approaches to Translocation Detection 177
Discordant Paired-End and Split Read-Based Analysis 177
Detection of Translocations and Inversions 177
RNA-Seq-Based Analysis 178
Translocation Detection in Clinical Practice 179
Laboratory Issues 180
Online Resources 181
Summary and Conclusion 182
References 182
11 Copy Number Variant Detection Using Next-Generation Sequencing 184
Overview of Copy Number Variation and Detection via Clinical Next-Generation Sequencing 185
Introduction 185
CNV Definition and Relationship to Other Classes of Structural Variation 185
Clinical CNV Screening and Potential for NGS-Based Discovery 186
Sources, Frequency, and Functional Consequences of Copy Number Variation in Humans 186
Mechanisms of CNV Generation 186
Frequency in the Human Genome 186
CNVs and Disease: Functional Consequences 187
CNV Detection in Clinical NGS Applications 189
Historical and Current Methods for Clinical Detection of CNVs 189
NGS in the Clinic: The Promise of Cost-Effective Comprehensive Mutation Testing 189
Targeted Sequencing of Candidate Genes 189
Exome Sequencing 191
Whole Genome Sequencing 191
Cell-Free NGS DNA Screening 192
Conceptual Approaches to NGS CNV Detection 192
Introduction 192
Discordant Mate Pair Methods 194
Depth of Coverage 195
SNP Allele Frequency 196
Split Reads and Local De Novo Assembly 197
Detection in the Clinic: Linking Application, Technical Approach, and Detection Methods 199
Targeted Gene Screening 199
Exome Sequencing for Unbiased Coding Variant Discovery 200
Cell-Free DNA 200
Whole Genome Sequencing and Emerging Technologies 201
Reference Standards 201
Genome Structural Variation Consortium Data Set 202
1000 Genomes Project Structural Variant Map Data Set 202
Orthogonal CNV Validation 203
Summary and Conclusion 203
References 203
Glossary 205
List of Acronyms and Abbreviations 206
III. Interpretation 208
12 Reference Databases for Disease Associations 210
Introduction 211
Identification and Validation of Human Variation 212
Methods for Identifying Human Variation 212
Targeting Known Sequences 212
Sequence Discovery 212
Sequencing and Identifying Differences Relative to a Reference 213
Understanding a Reference Assembly 213
Validation of Variant Calls 214
Identification of Common Variation 214
Overview 214
HapMap 215
1000 Genomes Project 216
NHLBI-ESP 217
Interpretation of Common Variation 218
Determining Association between Phenotype and Common Variation 218
Health-Related Phenotypes (GWAS) 218
Molecular Phenotypes (Gene Expression/GTex) 218
Inferring Lack of Causality Based on Allele Frequency 218
Defining Diseases and Phenotypes 218
UMLS and MedGen 219
OMIM® 219
Orphanet 219
Human Phenotype Ontology 220
Combining Phenotypic Features 220
Representation of Variation Data in Public Databases 220
Archives of Variants 220
dbSNP 220
dbVar 221
Archives of Variant/Phenotype Relationships 221
dbGaP 221
ClinVar 223
OMIM 223
DECIPHER 223
COSMIC 223
PubMed/PubMedCentral 224
Data Access and Interpretation 224
By One or More Genomic Locations 224
By Gene 227
By Condition or Reported Phenotype 227
By Attributes of a Particular Variant 227
Variants in the ACMG Incidental Findings Gene List 228
Determination of Variant Pathogenicity 229
Expert Panels and Professional Guidelines 229
ClinGen: A Centralized Resource of Clinically Annotated Genes and Variants 230
Standard Setting for Clinical Grade Databases 230
Global Data Sharing 231
GA4GH and the Beacon Project 231
Conclusion 232
References 232
List of Acronyms and Abbreviations 234
13 Reporting of Clinical Genomics Test Results 236
Introduction 238
Components of the Written NGS Report 238
Patient Demographics and Indication for Testing 238
Summary Statement of Test Interpretation 238
Variants That May Explain the Patient’s Phenotype 239
Gene Name and Transcript Number 239
Variant Nomenclature and Zygosity 240
Variant Classification and Supporting Evidence 240
Mutation Effect 240
Online Mutation Databases 240
Medical and Scientific Literature 241
Population Frequency 241
Computational Prediction Programs 241
Evolutionary Conservation 242
Protein Structure and Functional Domains 242
Functional Studies 242
Results of Familial Testing 242
Confirmation of Variants by a Secondary Method 242
Interpretation of the Test Result 242
Recommendations 243
Incidental or Secondary Findings 243
Technical Information About the Assay Performed 244
Methodology and Platform 244
Types of Mutations Detected by the Assay 244
Data Analysis and Variant Interpretation Algorithms 245
Depth of Coverage and Areas of No/Low Coverage 245
Analytical Sensitivity and Specificity 245
Clinical Sensitivity and Specificity 245
Additional Test Limitations 246
Disclaimer about FDA Approval 246
Signature of the Person Approving the Report 246
Beyond the Written Report: Other NGS Reporting Issues to Consider 246
Providing Raw Data to Clinicians and Patients 246
Reanalysis and Reinterpretation of NGS Data 247
Data Storage 247
Conclusion 247
References 247
List of Acronyms and Abbreviations 248
14 Reporting Software 250
Introduction 251
Clinical Genomic Test Order Entry 251
Laboratory Information Management Systems (LIMS) Tracking 252
Analytics: From Reads to Variant Calls 252
Analytical Validation 253
Provenance Tracking and Versioning 253
Pipeline Orchestration and Management 253
Analytics: Variant Annotation and Classification 254
Variant Interpretation 255
Final Report Transmission to the EMR 255
Leveraging Standards in Clinical Genomics Software Systems 256
Regulatory Compliance 256
Support Personnel 256
Conclusion 257
References 257
List of Acronyms and Abbreviations 258
15 Constitutional Diseases: Amplification-Based Next-Generation Sequencing 260
Introduction 260
Disease-Targeted Sequencing 260
Target Enrichment 261
Multigene Panel Validation 262
Select Genes 263
Design and Inspect Primers for Each Exon 263
Run Validation Samples 263
Bioinformatics 264
Clinical Workflow 264
Concurrent Testing 264
Bioinformatics and Data Interpretation 264
Conclusion 266
Advantages and Disadvantages of Amplification-Based NGS 266
Future Directions 267
References 267
List of Acronyms and Abbreviations 268
16 Targeted Hybrid Capture for Inherited Disease Panels 270
Introduction 271
Inherited Cardiomyopathies 271
Costello Syndrome 272
Hereditary Hearing Loss 272
Evolution of Medical Sequencing in Molecular Diagnostics 272
Target Selection Using Hybridization-Based Capture 274
Design and Implementation of Targeted Hybridization-Based Capture Panels 276
Technical Design Considerations 276
Determining the ROI 276
Ensuring Adequate Coverage Across the Entire ROI 276
Sequencing Regions of Increased or Decreased GC Content 277
Sequencing Regions with High Sequence Homology 278
Operational Considerations: Workflow, Cost and TAT 278
Workflow 278
Sequencing Cost 279
Cost-Reduction Measures 279
Factors Impacting the Ability to Batch and Pool Samples 279
Automation 279
Turnaround Time 280
Impact of the Type of Sequencing Machine 280
Targeted Hybrid Capture: Analytical Sensitivity Across the Variant Spectrum 280
Targeted Hybrid Capture: Selecting a Panel for Constitutional Diseases 281
Gene Panel Testing Strategy 281
Anticipating Technical Limitations 282
Regions with Low Coverage 282
GC-Rich or Repetitive Regions 282
Genes with Homology to Other Loci 282
Anticipating Interpretive Challenges: Impact of Panel Size on Variant Interpretation 282
Disease-Targeted Gene Panels: Comparison with other Sequencing Strategies 283
Whole Genome Sequencing 283
Whole Exome Sequencing 284
Amplification-Based Capture Methods 284
Other Target Selection Methods 284
Applications in Clinical Practice: Lessons Learned 285
Benefits of Targeted NGS Capture Panels 285
Inherited Cardiomyopathies 285
Hearing Loss and Related Disorders 286
Challenges 286
Conclusion and Outlook 286
References 287
17 Constitutional Disorders: Whole Exome and Whole Genome Sequencing 290
Introduction 292
Historical Perspective 293
Early Chromosomal Studies 294
Genomic-Based Studies: Genetic Markers 294
The Microarray 294
The GWAS Era 295
The Human Genome Project 295
Modern Sequencing Technologies 295
Genomic Sequencing 295
Advantages of Genomic Sequencing 295
Disadvantages of Genomic Sequencing 296
Comparison: Exomes Versus Genomes 296
What Regions Are Targeted/Covered? 296
Depth of Coverage 297
Type of Variants Detected 297
Resource-Based Considerations 297
Analyzing Individual and Multiple Data Sets for Causal Mutation Discovery 298
Phenotypically Similar Unrelated Probands 298
The Continued Importance of Clinical Analyses in the Era of Genomic Sequencing 299
Familial Studies 300
Recessive Diseases 300
De Novo Mutations 301
Using Databases of Population Variation 301
Issues and Concerns with the Use of Population Variation Databases to Filter Genomic Data Sets 301
Penetrance and Expressivity 302
The Accuracy and Reproducibility of Databases 302
Incorporation of Pathway-Related Data 303
Recognizing and Managing Artifacts 303
The Necessity of Independent Validation 304
Functional Interpretation of Variants 304
Combinatorial Approaches 305
Clinical Genomic Sequencing 306
Determining the Optimal Scope of Genetic/Genomic Investigations 306
Clinical Utility: Translating Genomic Knowledge from Rare Disease Research to more General Health Care Situations 307
The Clinical Timeline 307
Integrating the Management of Additional Genomic Information 307
Managing the Data Load in Clinical Scenarios 308
Consequences of Genomic Sequencing 309
Genetic Counseling and Ethical Issues 310
Conclusion and Future Directions 310
Acknowledgment 311
References 311
Glossary 315
18 Somatic Diseases (Cancer): Amplification-Based Next-Generation Sequencing 316
Introduction 317
NGS Technologies 317
Pyrosequencing-Based NGS: Roche 454 Genome Sequencer 317
Reversible Dye-Terminator-Based NGS: Illumina HiSeq and MiSeq Systems 318
Ion Semiconductor-Based NGS: Life Technology PGM and Proton Systems 319
Sequencing by Ligation-Based NGS: Life Technology ABI SOLiD Sequencer 320
Amplification-Based NGS Technologies 321
DNA Sequencing 322
Targeted DNA Analysis Using Multiplex Amplification 322
Targeted DNA Analysis Using Single-Plex Amplification 323
Targeted DNA Analysis Using Targeted Capture Followed by Multiplex Amplification 323
General Considerations 324
RNA Sequencing 324
Targeted RNA Analysis by Multiplex Amplification 324
Targeted RNA Analysis by Single-Plex Amplification 325
Targeted RNA Analysis Using Targeted Capture Followed by Multiplex Amplification 325
Methylation Analysis 326
Advantages and Disadvantages of Amplification-Based NGS 326
Clinical Application of Amplification-Based NGS in Cancer 327
Sample Requirements 328
DNA/RNA Extraction and Quality Control 328
Cancer-Specific Targeted Panels 329
AmpliSeq™ Cancer Hotspot Panel v2 330
Ion AmpliSeq™ Comprehensive Cancer Panel 330
AmpliSeq Custom Cancer Panels 331
Ion AmpliSeq™ RNA Cancer Panels 332
RainDance ONCOSeq™ Panel 332
Illumina TruSeq Amplicon Cancer Panel 332
Data Analysis 333
Interpretation and Reporting 334
Challenges and Perspectives 335
References 336
19 Targeted Hybrid-Capture for Somatic Mutation Detection in the Clinic 340
Introduction 341
Clinical Utility of Somatic Mutation Detection in Cancer 341
Description of Hybridization-Based Methodology 342
Solid-Phase Versus In-Solution Phase Capture 342
Comparison of In-Solution Hybridization Capture-Based and Amplification-Based Targeted Enrichment Methods for Molecular Onc... 343
Utility of Targeted Hybrid Capture 345
Analysis of Large Number of Genes Involved in Cancer 345
Applications for Precious Samples 345
Amenable to Multiplexing 345
Detection of a Full Range of Mutations 347
Detection of Structural Rearrangements (Translocations, Inversions, and Indels) 347
Copy Number Variation (CNV) Detection 350
Cost-Effectiveness 351
High Depth of Coverage 351
NGS in a Clinical Laboratory Setting 352
Design of the Clinical Assay 352
Specimen Requirements for Somatic Variant Detection 352
Pathologic Assessment 352
Reportable Range 353
Genetic Targets 353
QC Metrics 353
Validation 356
Conclusion 357
References 357
20 Somatic Diseases (Cancer): Whole Exome and Whole Genome Sequencing 362
Introduction to Exome and Genome Sequencing in Cancer 363
Interpretative Considerations in Exome and Genome Cancer Sequencing 363
Spectrum of Somatic Mutations in Cancer 363
Codon Level Mutations 364
Exon Level Mutations 364
Gene Level Mutations 365
Chromosome Level Mutations 365
Paired Tumor–Normal Testing 366
Tumor–Normal Comparison for Somatic Mutational Status 367
Determination of Somatic Status Without Paired Normal Tissue 367
Variants of Unknown Significance 368
General Categories of VUS 369
Statistical Models of Mutation Effect 369
Pathway Analysis 370
Driver Mutation Analysis 370
Clonal Architecture Analysis 370
Analytic Considerations for Exome and Genome Sequencing in Cancer 371
Specimen Requirements 371
Limitations 371
Decreased Depth of Coverage, Sensitivity, and Specificity 371
Advantages 373
Validation of a Single Assay 373
Evaluate Many Genes Simultaneously from One Sample 374
Improved Copy Number Variant Detection 374
Improved SV Detection—Genomes 375
Summary 375
References 376
IV. Regulation, Reimbursement, and Legal Issues 380
21 Assay Validation 382
Introduction 383
NGS Workflow 383
The Regulatory and Professional Framework for Assuring Quality 386
Assay Validation 386
Accuracy 388
Precision 389
Analytical Sensitivity and Analytical Specificity 390
Reportable and Reference Ranges 391
Quality Control 391
Reference Materials 392
Conclusion 392
Acknowledgment 393
References 393
List of Acronyms and Abbreviations 395
22 Regulatory Considerations Related to Clinical Next Generation Sequencing 396
Introduction 397
Regulatory Standards 397
FDA Oversight of Clinical NGS 398
Total Quality Management: QC 400
Preanalytic Variables 400
In Traditional Tests Not Shared by NGS 400
In Common with Traditional Tests 400
Unique to NGS 401
Analytic Variables 401
Sequencing Platform 402
Wet-Bench Procedures 402
Accuracy 402
Precision 402
Analytic Sensitivity and Analytic Specificity 402
Reportable Range and Reference Range 403
Sequence Verification 403
Specimen Provenance 403
Bioinformatic Pipeline 403
Independent Evaluation of the Different Classes of Mutations 403
Versioning and Revalidation 403
“Clinical Grade” Databases and Reference Materials 404
Postanalytic Variables 404
Total Quality Management: QA 405
Objectives of the QA Program 405
Proficiency Testing 405
Sample Exchange Programs 406
Analyte-Specific Versus Methods-Based PT 406
Cell Lines 406
Comprehensive PT Challenges Versus In Silico PT Challenges 407
Conclusion 407
References 408
23 Genomic Reference Materials for Clinical Applications 412
Introduction 412
Challenges in Developing a Whole Genome Reference Material 412
Genome in a Bottle Consortium 413
Reference Material Selection and Design 413
Reference Material Characterization 415
Bioinformatics, Data Integration, and Data Representation 415
Data Representation 416
Performance Metrics and Figures of Merit 416
Reference Data 418
Other Reference Materials for Genome-Scale Measurements 419
Microbial Genome RMs 419
Gene Expression RMs 419
Conclusion 420
References 420
24 Ethical Challenges to Next-Generation Sequencing 422
Introduction 423
Respect for Autonomy 425
Beneficence/Nonmaleficience 425
Justice 426
Conclusion 426
Challenging Existing Frameworks 427
Diagnostics Versus Screening 427
Research and Clinical Care 428
Individuals and Families 429
“What to Disclose” Is Becoming “What Not to Disclose” 429
Notifying of Results 430
Introduction 430
Different Kinds of Results 430
Research Results Versus Clinical Results 430
Raw Data 431
Probabilistic and Susceptibility Information 431
Variants of Unknown Significance (VUS) 431
Incidental Findings 431
Changing Status 432
How Do We Categorize Which Results to Return? 432
Analytic Validity 432
Clinical Validity 433
Clinical Utility 433
Personal Utility 434
ELSI (Ethical, Legal and Social Implications) 435
Recommendations 436
Recommendations 439
Privacy and Confidentiality 440
Introduction 440
Concepts 441
Data Protection Methods 441
Data Environment 442
Untrustworthy People 442
Reidentification 442
Required/Permitted Sharing 443
Recommendations 444
Informed Consent 444
Introduction 444
Recommendations 446
Balance the Amount of Information with Patient Initiative 446
The Right to Know and the Right Not to Know 447
Negotiation of Clinical and Personal Risks and Benefits 448
Evolving Results 448
Counseling 448
Transparency 449
Conclusion 449
References 449
Glossary 452
List of Acronyms and Abbreviations 453
25 Legal Issues 454
Introduction 454
Patent Overview 455
History of Gene Patents 455
Arguments for and Against Gene Patents 457
Important Legal Cases 457
Implication of Recent Court Decisions for Genetic Testing 462
Genetic Information Nondiscrimination Act 462
References 464
26 Billing and Reimbursement 466
Introduction 467
Insurance Payers 467
Reimbursement Processes 467
Reimbursement Rate 467
Diagnosis and Procedure Codes 469
Predetermination of Coverage and Benefits 471
Test Design Factors That Impact Reimbursement 471
Patient Protection and Affordable Care Act 473
Entities Focused on Healthcare Expenditures 473
Accountable Care Organizations 474
Health Outcomes 474
Cost Structure 475
Summary 475
References 476
Glossary 476
List of Acronyms and Abbreviations 477
Index 478