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.

ABI SOLiD™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.



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Further Reading

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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]