Next-generation DNA sequencing (NGS): Principles and clinical applications
- Peter J Hulick, MD, MMSc, FACMG
Peter J Hulick, MD, MMSc, FACMG
- Medical Director, Center for Medical Genetics
- NorthShore University HealthSystem
- Clinical Assistant Professor, University of Chicago, Pritzker School of Medicine
Technologies for sequencing DNA have improved dramatically, to the point that it has become practical to sequence an individual's entire genome. Next-generation sequencing (NGS) is a type of DNA sequencing technology that uses parallel sequencing of multiple small fragments of DNA to determine sequence. This "high-throughput" technology has allowed a dramatic increase in the speed (and a decrease in the cost) at which an individual's genome can be sequenced.
The ability to sequence an entire genome raises several challenging questions for the clinician, including the following:
●When should NGS be considered clinically?
●What is the best choice among several types of genetic testing available?
●What is the clinical significance of findings from sequencing of an entire genome?To continue reading this article, you must log in with your personal, hospital, or group practice subscription. For more information on subscription options, click below on the option that best describes you:
- Rizzo JM, Buck MJ. Key principles and clinical applications of "next-generation" DNA sequencing. Cancer Prev Res (Phila) 2012; 5:887.
- Bennett ST, Barnes C, Cox A, et al. Toward the 1,000 dollars human genome. Pharmacogenomics 2005; 6:373.
- Hong YC, Liu HM, Chen PS, et al. Hair follicle: a reliable source of recipient origin after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2007; 40:871.
- Thiede C, Prange-Krex G, Freiberg-Richter J, et al. Buccal swabs but not mouthwash samples can be used to obtain pretransplant DNA fingerprints from recipients of allogeneic bone marrow transplants. Bone Marrow Transplant 2000; 25:575.
- Rennert H, Leonard DG, Cushing M, et al. Avoiding pitfalls in bone marrow engraftment analysis: a case study highlighting the weakness of using buccal cells for determining a patient's constitutional genotype after hematopoietic stem cell transplantation. Cytotherapy 2013; 15:391.
- Easton DF, Pharoah PD, Antoniou AC, et al. Gene-panel sequencing and the prediction of breast-cancer risk. N Engl J Med 2015; 372:2243.
- King MC, Levy-Lahad E, Lahad A. Population-based screening for BRCA1 and BRCA2: 2014 Lasker Award. JAMA 2014; 312:1091.
- Walsh T, Casadei S, Lee MK, et al. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc Natl Acad Sci U S A 2011; 108:18032.
- Selkirk CG, Vogel KJ, Newlin AC, et al. Cancer genetic testing panels for inherited cancer susceptibility: the clinical experience of a large adult genetics practice. Fam Cancer 2014; 13:527.
- Rehm HL, Bale SJ, Bayrak-Toydemir P, et al. ACMG clinical laboratory standards for next-generation sequencing. Genet Med 2013; 15:733.
- Strom SP, Lee H, Das K, et al. Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory. Genet Med 2014; 16:510.
- Beck TF, Mullikin JC, NISC Comparative Sequencing Program, Biesecker LG. Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants. Clin Chem 2016; 62:647.
- Taylor JC, Martin HC, Lise S, et al. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nat Genet 2015; 47:717.
- Bamshad MJ, Ng SB, Bigham AW, et al. Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 2011; 12:745.
- Lupski JR, Reid JG, Gonzaga-Jauregui C, et al. Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy. N Engl J Med 2010; 362:1181.
- Ashley EA, Butte AJ, Wheeler MT, et al. Clinical assessment incorporating a personal genome. Lancet 2010; 375:1525.
- Rehm HL, Berg JS, Brooks LD, et al. ClinGen--the Clinical Genome Resource. N Engl J Med 2015; 372:2235.
- Van Driest SL, Wells QS, Stallings S, et al. Association of Arrhythmia-Related Genetic Variants With Phenotypes Documented in Electronic Medical Records. JAMA 2016; 315:47.
- Amendola LM, Jarvik GP, Leo MC, et al. Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium. Am J Hum Genet 2016; 98:1067.
- Biesecker LG, Green RC. Diagnostic clinical genome and exome sequencing. N Engl J Med 2014; 370:2418.
- Lee H, Deignan JL, Dorrani N, et al. Clinical exome sequencing for genetic identification of rare Mendelian disorders. JAMA 2014; 312:1880.
- Wright CF, Fitzgerald TW, Jones WD, et al. Genetic diagnosis of developmental disorders in the DDD study: a scalable analysis of genome-wide research data. Lancet 2015; 385:1305.
- Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013; 369:1502.
- Yang Y, Muzny DM, Xia F, et al. Molecular findings among patients referred for clinical whole-exome sequencing. JAMA 2014; 312:1870.
- Gilissen C, Hehir-Kwa JY, Thung DT, et al. Genome sequencing identifies major causes of severe intellectual disability. Nature 2014; 511:344.
- Tarailo-Graovac M, Shyr C, Ross CJ, et al. Exome Sequencing and the Management of Neurometabolic Disorders. N Engl J Med 2016; 374:2246.
- Kuehn BM. NIH's Undiagnosed Diseases Program expands: 6 new sites offer potential answers to more patients. JAMA 2014; 312:587.
- Lazaridis KN, Schahl KA, Cousin MA, et al. Outcome of Whole Exome Sequencing for Diagnostic Odyssey Cases of an Individualized Medicine Clinic: The Mayo Clinic Experience. Mayo Clin Proc 2016; 91:297.
- https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm585347.htm (Accessed on November 16, 2017).
- https://www.accessdata.fda.gov/cdrh_docs/reviews/DEN170058.pdf (Accessed on November 16, 2017).
- https://www.cms.gov/Newsroom/MediaReleaseDatabase/Press-releases/2017-Press-releases-items/2017-11-30-2.html (Accessed on December 04, 2017).
- Zehir A, Benayed R, Shah RH, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med 2017; 23:703.
- https://www.cms.gov/Medicare/Coverage/DeterminationProcess/downloads/id290.pdf (Accessed on December 04, 2017).
- https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/125514s024lbl.pdf (Accessed on November 27, 2017).
- Sintchenko V, Holmes EC. The role of pathogen genomics in assessing disease transmission. BMJ 2015; 350:h1314.
- Wilson MR, Naccache SN, Samayoa E, et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N Engl J Med 2014; 370:2408.
- Kalia SS, Adelman K, Bale SJ, et al. Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics. Genet Med 2017; 19:249.
- Vassy JL, Christensen KD, Schonman EF, et al. The Impact of Whole-Genome Sequencing on the Primary Care and Outcomes of Healthy Adult Patients: A Pilot Randomized Trial. Ann Intern Med 2017.
- Long EF, Ganz PA. Cost-effectiveness of Universal BRCA1/2 Screening: Evidence-Based Decision Making. JAMA Oncol 2015; 1:1217.
- Waddell N, Pajic M, Patch AM, et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 2015; 518:495.
- Goldfeder RL, Wall DP, Khoury MJ, et al. Human Genome Sequencing at the Population Scale: A Primer on High-Throughput DNA Sequencing and Analysis. Am J Epidemiol 2017; 186:1000.
- Association for Molecular Pathology v. Myriad Genetics, Inc. No. 12-398. 569 U.S ___ (2013). Text available at: http://www.supremecourt.gov/opinions/12pdf/12-398_1b7d.pdf
- TERMINOLOGY AND EVOLUTION OF TECHNOLOGIES
- TECHNICAL CONSIDERATIONS
- Source of DNA
- Whole genome, exome, or gene panel
- Mutation databases
- CLINICAL USE OF NEXT-GENERATION SEQUENCING
- Indications for NGS
- - Diagnosis of complex diseases
- - Cancer screening and management
- - Diagnosis of infections
- - Healthy people
- Genetic discrimination
- Disclosure of findings from genome sequencing
- PRACTICAL ISSUES
- Where to order
- Cost and turnaround time
- Insurance reimbursement