The challenges of sequencing by synthesis

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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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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