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The challenges of sequencing by synthesis

Nature Biotechnology volume 27, pages 1013–1023 (2009)Cite this article

Abstract

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to $100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

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Figure 1: Schemes for SBS.
Figure 2: Sample preparation includes arraying individual fragments of DNA on beads or other solid surface.
Figure 3: Surface chemistry.
Figure 4: Enzyme substrate system.
Figure 5: Read-length and phasing.

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Acknowledgements

This work was supported in part by the National Human Genome Research Institute, National Institutes of Health.

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Authors and Affiliations

  1. GE Healthcare Life Sciences, Piscataway, New Jersey, USA

    Carl W Fuller

  2. LI-COR Biosciences, Lincoln, Nebraska, USA

    Lyle R Middendorf

  3. Foundation for Applied Molecular Evolution, Alachua, Florida, USA

    Steven A Benner

  4. Harvard Medical School, Boston, Massachusetts, USA

    George M Church

  5. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA

    Timothy Harris

  6. University of California, San Diego, La Jolla, Californi, USA

    Xiaohua Huang

  7. Microchip Biotechnologies Inc., Dublin, California, USA

    Stevan B Jovanovich

  8. General Electric Global Research Center, Niskayuna, New York, USA

    John R Nelson

  9. National Human Genome Research Institute, NIH, Bethesda, Maryland, USA

    Jeffery A Schloss

  10. University of Wisconsin-Madison, Madison, Wisconsin, USA

    David C Schwartz

  11. Lehigh University, Bethlehem, Pennsylvania, USA

    Dmitri V Vezenov

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Contributions

C.W.F. and L.R.M. wrote this review, with additions and editorial assistance from S.A.B., G.M.C., T.H., X.H., S.B.J., J.R.N., D.C.S. and D.V.V., who contributed portions of the text and read drafts of the manuscript for accuracy. J.A.S. is the scientific manager of the NHGRI Sequencing Technology Development Program; he proposed the idea for the review, provided a forum to begin its formulation at a program meeting and read the manuscript for accuracy.

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Correspondence to Carl W Fuller.

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G.M.C. plays advisory roles at several companies relevant to this paper and these are listed at http://arep.med.harvard.edu/gmc/tech.html.

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Fuller, C., Middendorf, L., Benner, S. et al. The challenges of sequencing by synthesis. Nat Biotechnol 27, 1013–1023 (2009). https://doi.org/10.1038/nbt.1585

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