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  • Perspective
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The chemistry of next-generation sequencing

Nature Biotechnology volume 41, pages 1709–1715 (2023)Cite this article

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Abstract

The first large genome fully sequenced by next-generation sequencing (NGS) was that of a bacteriophage using sequencing by synthesis (SBS) as a paradigm. SBS in NGS is underpinned by ‘reversible-terminator chemistry’. To grow from proof of concept to being both affordable and practical, SBS needed to overcome a series of challenges, each of which required the invention of new chemistries. These included the design and synthesis of unnatural deoxynucleotide triphosphates (dNTPs), engineering a suitable polymerase, a new surface chemistry and an ingenious molecular solution to neutralize copying errors inherent to all polymerases. In this historical Perspective, we discuss how NGS was developed from Sanger sequencing, highlighting the chemistry behind this technology, which has impacted biology in unprecedented ways.

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Fig. 1: Schematic illustration of Sanger sequencing.
Fig. 2: Scheme of reversible-terminator chemistry.
Fig. 3: Schematic illustration of the active site within DNA polymerase.
Fig. 4: Schematic of the NGS workflow (part 1).
Fig. 5: Schematic of the NGS workflow (part 2).

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Acknowledgements

We thank all those whose intellectual and practical contributions have collectively enabled the development of NGS. The intellectual property underlying NGS is protected by a series of patents not discussed in this article. We thank J. Szostak, V. Ramakrishnan and J.-M. Lehn for fruitful discussions and our colleagues at the University of Chicago and the Institut Curie for their constructive comments. R.R. was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 647973), Foundation Charles Defforey-Institut de France and Ligue Contre le Cancer. Y.K. is supported by NIH grant 1DP1GM149751-01 and the Ono Pharma Foundation.

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Authors and Affiliations

  1. Institut Curie, CNRS, INSERM, PSL Research University, Equipe Labellisée Ligue Contre le Cancer, Paris, France

    Raphaël Rodriguez

  2. Department of Chemistry, the University of Chicago, Chicago, IL, USA

    Yamuna Krishnan

  3. The Neuroscience Institute, the University of Chicago, Chicago, IL, USA

    Yamuna Krishnan

  4. Institute of Biophysical Dynamics, the University of Chicago, Chicago, IL, USA

    Yamuna Krishnan

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  1. Raphaël Rodriguez
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R.R. and Y.K. contributed equally to this work.

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Correspondence to Raphaël Rodriguez or Yamuna Krishnan.

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Rodriguez, R., Krishnan, Y. The chemistry of next-generation sequencing. Nat Biotechnol 41, 1709–1715 (2023). https://doi.org/10.1038/s41587-023-01986-3

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