Next‐generation Sequencing in Clinical Molecular Diagnostics


Next‐generation sequencing is based upon the concept of massively parallel chemical reactions in which millions of independent DNA sequencing events occur simultaneously, and this is achieved using a relatively similar concept across all of the currently available platforms. Current clinical molecular genetics labs offer testing mainly for single‐gene defects and a few multigene panels. However, for whole‐exome and whole‐genome sequencing, as the cost per sample continues to fall, there is little question that the clinical application of this technology will soon become abundant in diagnostic labs. Before that happens, we will need to prepare ourselves to handle certain types of unexpected and potentially unwanted information, since this technology should prove to be of tremendous value for the practice of medicine in the future.

Key Concepts:

  • Next‐generation sequencing is based upon the concept of massively parallel chemical reactions in which millions of independent DNA sequencing events occur simultaneously.

  • There are several commercial vendors of next‐generation sequencing instruments, though the market is currently dominated by Roche, Illumina and Life Technologies.

  • Most clinical labs prefer to sequence multiple samples or many regions of the genome in one run, and there are several methods that can be used to accomplish this.

  • Next‐generation sequencing can also be used to effectively detect both large and small chromosome aberrations.

  • Next‐generation sequencing has the potential to increase the scope of tests for sequence variants in the areas of infectious disease testing and oncology.

  • Clinical laboratories are currently observing caution in the adoption of this new technology.

Keywords: sequencing; parallel; Sanger; bioinformatics; library; capture

Figure 1.

Roche 454 Genome Sequencer FLX System chemistry. (a) Emulsion‐based clonal amplification of adapter‐ligated DNA fragments on beads. (b) Deposition of single clonally amplified DNA beads into the PicoTiterPlate™ device. The well diameter of the PicoTiterPlate™ allows only one bead per well. (c) Smaller beads carrying enzymes necessary for the pyrosequencing reaction are added to all wells of the PicoTiterPlate™. (d) Pyrosequencing reaction of the FLX System. Nucleotides are sequentially flowed across the PicoTiterPlate™, generating a chemiluminescent signal that allows for base determination of each DNA capture bead in parallel. Reproduced by permission of Roche Diagnostics.

Figure 2.

Illumina 1G Genome Analyser chemistry. (a) A dilute concentration of single‐stranded adapter‐ligated fragments are bound to the inside surface of the flow cell. (b) Unlabelled nucleotides and DNA polymerase are added to initiate solid‐phase bridge amplification and form fragment clusters. (c) After one round of extension a complementary fragment is formed, also with one free terminus and opposite end attached to the slide. (d) The fragments are denatured and a new round of bridge amplification is initiated. (e) Upon the completion of several rounds of bridge amplification, several million dense clusters of clonal DNA fragments are generated in each channel of the flow cell. (f) To initiate the first sequencing cycle, all four fluorescently labelled reversible terminators are added simultaneously to the flow cell along with primers and DNA polymerase. Reproduced by permission of Illumina Inc.

Figure 3.

ABISOLiD™4‐colour ligation reaction chemistry. (a) Four dyes encode for 16 possible two‐base combinations. Each probe interrogates only the first two bases specifically. Multiple cycles of ligation, detection and cleavage are performed followed by (b) removal of the template and the addition of a ‘frameshift’ anchor primer 1 bp smaller than the primer from the previous round of ligation cycles. After five primer rounds of seven ligation cycles each, 35 bp have been interrogated twice. For example, the base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by primer number 3 in ligation cycle 1. Reproduced by permission of Applied Biosystems/Life Technologies.



Albert TJ, Molla MN, Muzny DM et al. (2007) Direct selection of human genomic loci by microarray hybridization. Nature Methods 4: 903–905.

Binladen J, Gilbert MT, Bollback JP et al. (2007) The use of coded PCR primers enables high‐throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS ONE 2: e197.

Campbell PJ, Stephens PJ, Pleasance ED et al. (2008) Identification of somatically acquired rearrangements in cancer using genome‐wide massively parallel paired‐end sequencing. Nature Genetics 40: 722–729.

Castle JC, Biery M, Bouzek H et al. (2010) DNA copy number, including telomeres and mitochondria, assayed using next‐generation sequencing. BMC Genetics 11: 244–254.

Chiang DY, Getz G, Jaffe DB et al. (2009) High‐resolution mapping of copy‐number alterations with massively parallel sequencing. Nature Methods 6: 99–103.

Daiger SP, Sullivan LS, Bowne SJ et al. (2010) Targeted high‐throughput DNA sequencing for gene discovery in retinitis pigmentosa. Advances in Experimental Medicine and Biology 664: 325–331.

Gharizadeh B, Herman ZS, Eason RG, Jejelowo O and Pourmand N (2006) Large‐scale pyrosequencing of synthetic DNA: a comparison with results from Sanger dideoxy sequencing. Electrophoresis 27: 3042–3047.

Gnirke A, Melnikov A, Maguire J et al. (2009) Solution hybrid selection with ultra‐long oligonucleotides for massively parallel targeted sequencing. Nature Biotechnology 27: 182–189.

Gowrisankar S, Lerner‐Ellis JP, Cox S et al. (2010) Evaluation of second‐generation sequencing of 19 dilated cardiomyopathy genes for clinical applications. Journal of Molecular Diagnostics 12: 818–827.

Hodges E, Xuan Z, Balija V et al. (2007) Genome‐wide in situ exon capture for selective resequencing. Nature Genetics 39: 1522–1527.

Krishnakumar S, Zheng J, Wilhelmy J et al. (2008) A comprehensive assay for targeted multiplex amplification of human DNA sequences. Proceedings of the National Academy of Sciences of the USA 105: 9296–9301.

Leary RJ, Kinde I, Diehl F et al. (2010) Development of personalized tumor biomarkers using massively parallel sequencing. Science Translational Medicine 2: 20ra14.

Lupski JR, Reid JG, Gonzaga‐Jauregui C et al. (2010) Whole‐genome sequencing in a patient with charcot marie tooth neuropathy. New England Journal of Medicine 362: 1181–1191.

Margulies M, Egholm M, Altman WE et al. (2005) Genome sequencing in microfabricated high‐density picolitre reactors. Nature 437: 376–380.

Meyer M, Stenzel U, Myles S, Prufer K and Hofreiter M (2007) Targeted high‐throughput sequencing of tagged nucleic acid samples. Nucleic Acids Research 35: e97.

Ng SB, Bigham AW, Buckingham KJ et al. (2010) Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nature Genetics 42: 790–793.

Okou DT, Steinberg KM, Middle C et al. (2007) Microarray‐based genomic selection for high‐throughput resequencing. Nature Methods 4: 907–909.

Ondov BD, Varadarajan A, Passalacqua KD and Bergman NH (2008) Efficient mapping of Applied Biosystems SOLiD sequence data to a reference genome for functional genomic applications. Bioinformatics 24: 2776–2777.

Palacios G, Druce J, Du L et al. (2008) A new arenavirus in a cluster of fatal transplant‐associated diseases. New England Journal of Medicine 358: 991–998.

Porreca GJ, Zhang K, Li JB et al. (2010) Multiplex amplification of large sets of human exons. Nature Methods 4: 931–936.

Raca G, Jackson C, Warman B, Bair T and Schimmenti LA (2010) Next generation sequencing in research and diagnostics of ocular birth defects. Molecular Genetics and Metabolism 100: 184–192.

Richards CS, Bale S, Bellissimo DB et al. (2008) ACMG recommendations for standards for interpretation and reporting of sequence variations: revisions 2007. Genetics in Medicine 10: 294–300.

Tarpey PS, Smith R, Pleasance E et al. (2009) A systematic, large‐scale resequencing screen of X‐chromosome coding exons in mental retardation. Nature Genetics 41: 535–543.

Tewhey R, Warner JB, Nakano M et al. (2009) Microdroplet‐based PCR enrichment for large‐scale targeted sequencing. Nature Biotechnology 27: 1025–1031.

Vasta V, Ng SB, Turner EH, Shendure J and Hahn SH (2009) Next generation sequence analysis for mitochondrial disorders. Genome Medicine 1: 100.

Wheeler M, Pavlovic A, DeGoma E et al. (2009) A new era in clinical genetic testing for hypertrophic cardiomyopathy. Journal of Cardiovascular Translational Research 2: 381–391.

Yoon S, Xuan Z, Makarov V, Ye K and Sebat J (2009) Sensitive and accurate detection of copy number variants using read depth of coverage. Genome Research 19: 1586–1592.

Further Reading

Bainbridge MN, Wiszniewski W, Murdock DR et al. (2011) Whole‐genome sequencing for optimized patient management. Science Translational Medicine 3(87): 87re3.

ten Bosch JR and Grody WW (2008) Keeping up with the next generation: massively parallel sequencing in clinical diagnostics. Journal of Molecular Diagnostics 10(6): 484–492.

Metzker ML (2010) Sequencing technologies – the next generation. Nature Reviews Genetics 11(1): 31–46.

Roach JC, Glusman G, Smit AF et al. (2010) Analysis of genetic inheritance in a family quartet by whole‐genome sequencing. Science 328(5978): 636–639.

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ten Bosch, John R, Deignan, Joshua L, and Grody, Wayne W(Sep 2011) Next‐generation Sequencing in Clinical Molecular Diagnostics. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0022483]