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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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References

  1. Schloss, J. How to get genomes at one ten-thousandth the cost. Nat. Biotechnol. 26, 1113–1115 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Dohm, J.C., Lottaz, C., Borodina, T. & Himmelbauer, H. Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res. 36, e105, 2008.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Craig, D.W. et al. Identification of genetic variants using bar-coded multiplexed sequencing. Nat. Methods 5, 887–893 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Morozova, O. & Marra, M.A. Applications of next-generation sequencing technologies in functional genomics. Genomics 92, 255–264 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Mardis, E. Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet. 9, 387–402 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Mardis, E.R. The impact of next-generation sequencing technology on genetics. Trends Genet. 24, 133–141 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Chi, K.R. The year of sequencing. Nat. Methods 5, 11–14 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Marguerat, S., Wilhelm, B.T. & Bähler, J. Next-generation sequencing: applications beyond genomes. Biochem. Soc. Trans. 36, 1091–1096 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Smith, D.R. et al. Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res. 18, 1638–1642 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Quinn, N.L. et al. Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome. BMC Genomics 9, 404 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Sarin, S. et al. Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nat. Methods 5, 865–867 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Holt, R.A. & Jones, S.J.M. The new paradigm of flow cell sequencing. Genome Res. 18, 839–846 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Rothberg, J.M. & Leamon, J.H. The development and impact of 454 sequencing. Nat. Biotechnol. 26, 1117–1124 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Kahvejian, A., Quackenbush, J. & Thompson, J.F. What would you do if you could sequence everything? Nat. Biotechnol. 26, 1125–1133 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Shendure, J. & Hanlee, J. Next-generation DNA sequencing. Nat. Biotechnol. 26, 1135–1145 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Branton, D. et al. Nanopore sequencing. Nat. Biotechnol. 26, 1146–1153 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Levene, M.J. et al. Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682–686 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Korlach, J. et al. Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. Proc. Natl. Acad. Sci. USA 105, 1176–1181 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Williams, J. System and method for nucleic acid sequencing by polymerase synthesis. US patent application US20030194740 (2003).

    Google Scholar 

  21. Willams, J. & Anderson, J. Field-switch sequencing. US patent application US20050266456 (2005).

    Google Scholar 

  22. Hardin, S., Gao, X., Briggs, J., Willson, R. & Tu, S.C. Real-time sequence determination. US patent application US20030064366 (2003).

    Google Scholar 

  23. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Harris, T.D. et al. Single-molecule DNA sequencing of a viral genome. Science 320, 106–109 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Milton, J. et al. Modified nucleotides (for polynucleotide sequencing). World and US patent application WO2004/018497, US2007/0166705 (2004).

    Google Scholar 

  26. Bentley, D.R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Turcatti, G., Romieu, A., Fedurco, M. & Tairi A.P. A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. Nucleic Acids Res. 36, e25 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Guo, J. et al. Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc. Natl. Acad. Sci. USA 105, 9145–9150 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ju, J. et al. Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proc. Natl. Acad. Sci. USA 103, 19635–19640 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Seo, T.S. et al. Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides. Proc. Natl. Acad. Sci. USA 102, 5926–5931 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wu, W. et al. Termination of DNA synthesis by N6-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates. Nucleic Acids Res. 35, 6339–6349 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. McKernan, K., Blanchard, A., Kotler, L. & Costa, G. Reagents, methods and libraries for bead-based sequencing. PCT patent application WO2006084132 (2007).

    Google Scholar 

  33. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Fuller, C.W. & Nelson, J.R. Method for nucleic acid analysis. PCT patent application WO2005123957 (2005).

    Google Scholar 

  35. Fuller, C.W. Rapid parallel nucleic acid analysis. US patent application US20060051807 (2006).

    Google Scholar 

  36. Ronaghi, M., Uhlén, M. & Nyrén, P. A sequencing method based on real-time pyrophosphate. Science 281, 363–365 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Scheibye-Alsing, K. et al. Sequence assembly. Comput. Biol. Chem. 33, 121–136 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. Trapnell, C. & Salzberg, S.L. How to map billions of short reads onto genomes. Nat. Biotechnol. 27, 455–457 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chetverina, H.V. & Chetverin, A.B. Cloning of RNA molecules in vitro. Nucleic Acids Res. 21, 2349–2353 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Church, G.M. Replica amplification of nucleic acid arrays. US patent 6,432,360 (2002).

    Google Scholar 

  41. Dressman, D., Yan, H., Traverso, G., Kinzler, K.W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. USA 100, 8817–8822 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Embleton, M.J., Gorochov, G., Jones, P.T. & Winter, G.P. In situ recombinant PCR within single cells US Patent 5,830,663 (1998).

    Google Scholar 

  43. Griffiths, A. & Tawfik, D. In vitro sorting method. US patent 6,489,103 (2002).

    Google Scholar 

  44. Holliger, P. & Ghadessy, F. Emulsion compositions. US patent 7,429,467 (2008).

    Google Scholar 

  45. Brenner, S. et al. In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs. Proc. Natl. Acad. Sci. USA 97, 1665–1670 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Adessi, C., Kawashima, E., Mayer, P., Mermod, J.J. & Turcatti, G. Methods of nucleic acid amplification and sequencing. PCT patent application WO2000018957 (2000).

    Google Scholar 

  47. Boles, T.C., Kron, S.J. & Adams, C.P. Nucleic acid-containing polymerizable complex. US patent 5,932,711 (1999).

    Google Scholar 

  48. Zhang, K. et al. Long-range polony haplotyping of individual human chromosome molecules. Nat. Genet. 38, 382–387 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Zhang, K. et al. Sequencing genomes from single cells via polymerase clones. Nat. Biotechnol. 24, 680–686 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    Article  CAS  PubMed  Google Scholar 

  51. Li, M., Diehl, F., Dressman, D., Vogelstein, B. & Kinzler, K.W. BEAMing up for detection and quantification of rare sequence variants. Nat. Methods 3, 95–97 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Pihlak, A. et al. Rapid genome sequencing with short universal tiling probes. Nat. Biotechnol. 26, 676–684 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Liu, D., Daubendiek, S.L., Zillman, M.A., Ryan, K. & Kool, E.T. Rolling circle DNA synthesis: small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118, 1587–1594 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Baner, J., Nilsson, M., Mendel-Hartvig, M. & Landegren, U. Signal amplification of padlock probes by rolling circle replication. Nucleic Acids Res. 26, 5073–5078 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Fire, A. & Xu, S.Q. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92, 4641–4645 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Blanco, L. et al. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940 (1989).

    CAS  PubMed  Google Scholar 

  57. Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Lizardi, P.M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).

    Article  CAS  PubMed  Google Scholar 

  59. Ramanathan, A. et al. An integrative approach for the optical sequencing of single DNA molecules. Anal. Biochem. 330, 227–241 (2004).

    Article  CAS  PubMed  Google Scholar 

  60. Parameswaran, P. et al. A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res. 35, e130 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Albert, T.J. et al. Direct selection of human genomic loci by microarray hybridization. Nat. Methods 4, 903–905 (2007).

    Article  CAS  PubMed  Google Scholar 

  62. Porreca, G.J. et al. Multiplex amplification of large sets of human exons. Nat. Methods 4, 931–936 (2007).

    Article  CAS  PubMed  Google Scholar 

  63. Kim, J.B. et al. Polony multiplex analysis of gene expression (PMAGE) in mouse hypertrophic cardiomyopathy. Science 316, 1481–1484 (2007).

    Article  CAS  PubMed  Google Scholar 

  64. Williams, J.G. et al. An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces. Nucl. Acids Res. 36, e121 (2008).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  65. Barbee, K.D. & Huang, X. Magnetic assembly of high-density DNA arrays for genomic analyses. Anal. Chem. 80, 2149–2154 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Brenner, S. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat. Biotechnol. 18, 630–634 (2000).

    Article  CAS  PubMed  Google Scholar 

  67. Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R. & Waggoner, A.S. Cyanine dye labeling reagents containing isothiocyanate groups. Cytometry 10, 11–19 (1989).

    Article  CAS  PubMed  Google Scholar 

  68. Sood, A. et al. Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays. J. Am. Chem. Soc. 127, 2394–2395 (2005).

    Article  CAS  PubMed  Google Scholar 

  69. Prober, J.M. et al. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238, 336–341 (1987).

    Article  CAS  PubMed  Google Scholar 

  70. Langer, P.R., Waldrop, A.A. & Ward, D.C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633–6637 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Wu, W. et al. Termination of DNA synthesis by N6-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates. Nucleic Acids Res. 35, 6339–6349 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Canard, B. & Sarfati, R.S. DNA polymerase fluorescent substrates with reversible 3′-tags. Gene 148, 1–6 (1994).

    Article  CAS  PubMed  Google Scholar 

  73. Tabor, S. & Richardson, C.C. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc. Natl. Acad. Sci. USA 92, 6339–6343 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Doublié, S., Tabor, S., Long, A.M., Richardson, C.C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251–258 (1998).

    Article  PubMed  Google Scholar 

  75. Kumar, S. et al. Terminal phosphate labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases. Nucleosides Nucleotides Nucleic Acids 24, 401–408 (2005).

    Article  CAS  PubMed  Google Scholar 

  76. Mulder, B.A. et al. Nucleotide modification at the gamma-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase. Nucleic Acids Res. 33, 4865–4873 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Williams, J.G.K., Anderson, J.P., Urlacher, T.M. & Steffens, D.L. Mutant polymerases for sequencing and genotyping. US patent application US20070048748 (2007).

    Google Scholar 

  78. Williams, J.G.K. Polymerases with charge-switch activity and methods of generating such polymers. US published patent application US20040259082 (2004).

    Google Scholar 

  79. Korlach, J. et al. Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. Nucleosides Nucleotides Nucleic Acids 27, 1072–1083 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Reynolds, B., Miller, R., Williams, J.G. & Anderson, J.P. Synthesis and stability of novel terminal phosphate-labeled nucleotides. Nucleosides Nucleotides Nucleic Acids 27, 18–30 (2008).

    Article  CAS  PubMed  Google Scholar 

  81. Steffens, D.L. & Williams, J.G. Efficient site-directed saturation mutagenesis using degenerate oligonucleotides. J. Biomol. Tech. 18, 147–149 (2007).

    PubMed  PubMed Central  Google Scholar 

  82. Ryu, J., Hong, S.S., Horn, B.K.P., Freeman, D.M. & Mermelstein, M.S. Multibeam interferometric illumination as the primary source of resolution in optical microscopy. Appl. Phys. Lett. 88, 171112 (2006).

    Article  CAS  Google Scholar 

  83. Mico, V. et al. Transverse resolution improvement using rotating-grating time-multiplexing approach. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 25, 1115–1129 (2008).

    Article  PubMed  Google Scholar 

  84. Pierce, J.R. An Introduction to Information Theory, edn. 2 (Dover Press, Mineola, NY, 1980).

    Google Scholar 

  85. Lundstrom, M. Moore's law forever? Science 299, 210–211 (2001).

    Article  Google Scholar 

  86. Stelzer, E.H.K. Beyond the diffraction limit? Nature 417, 806–807 (2002).

    Article  CAS  PubMed  Google Scholar 

  87. Lloyd, S. Ultimate physical limits to computation. Nature 406, 1047–1054 (2000).

    Article  CAS  PubMed  Google Scholar 

  88. Lloyd, S., Giovannetti, V. & Maccone, L. Physical limits to communication. Phys. Rev. Lett. 93, 100501–100504 (2004).

    Article  PubMed  CAS  Google Scholar 

  89. Stelzer, E.H.K. & Grill, S. The uncertainty principle applied to estimate focal spot dimensions. Opt. Commun. 173, 51–56 (2000).

    Article  CAS  Google Scholar 

  90. Moerner, W.E. & Fromm, D.P. Methods of single-molecule fluorescence spectroscopy and microscopy. Rev. Sci. Inst. 74, 3597–3619 (2003).

    Article  CAS  Google Scholar 

  91. Basché, Th., Ambrose, W.P. & Moerner, W.E. Optical spectra and kinetics of single impurity molecules in a polymer: spectral diffusion and persistent spectral hole burning. J. Opt. Soc. Am. B 9, 829–836 (1992).

    Article  Google Scholar 

  92. Stevenson, C.L. & Winefordner, J.D. Estimating detection limits in ultratrace analysis. Part I: The variability of estimated detection limits. Applied Spect. 45, 1217–1224 (1991).

    Article  CAS  Google Scholar 

  93. Stevenson, C.L. & Winefordner, J.D. Estimating detection limits in ultratrace analysis. Part II: Detecting and counting atoms and molecules. Applied Spect. 46, 407–419 (1992).

    Article  CAS  Google Scholar 

  94. Stevenson, C.L. & Winefordner, J.D. Estimating detection limits in ultratrace analysis. Part III: Monitoring atoms and molecules with laser-induced fluorescence. Applied Spect. 46, 715–724 (1992).

    Article  CAS  Google Scholar 

  95. Landauer, R. Minimal energy requirements in communication. Science 272, 1914–1918 (1996).

    Article  CAS  PubMed  Google Scholar 

  96. Zurek, W.H. Thermodynamic cost of computation, algorithmic complexity and the information metric. Nature 341, 119–124 (1989).

    Article  Google Scholar 

  97. Simon, S.H., Moustakas, A.L., Stoytchev, M. & Safar, H. Communication in a disordered world. Phys. Today 54, 38–43 (2001).

    Article  Google Scholar 

  98. Méray, L. & Demény, O. Detection limit and decision thresholds in spectrometry. Appl. Spect. 55, 1102–1108 (2001).

    Article  Google Scholar 

  99. Lachmann, M., Newman, M.E.J. & Moore, C. The physical limits of communication. Am. J. Phys. 72, 1290–1293 (2004).

    Article  Google Scholar 

  100. Gordon, J.M. & Feuermann, D. Optical performance at the thermodynamic limit with tailored imaging designs. Appl. Opt. 44, 2327–2331 (2005).

    Article  PubMed  Google Scholar 

  101. Sheehan, P.E. & Whitman, L.J. Detection limits for nanoscale biosensors. Nano Lett. 5, 803–807 (2005).

    Article  CAS  PubMed  Google Scholar 

  102. Prummer, M. Sick., B. Renn, A. & Wild, U.P. Multiparameter microscopy and spectroscopy for single-molecule analytics. Anal. Chem. 76, 1633–1640 (2004).

    Article  CAS  PubMed  Google Scholar 

  103. Hubaux, A. & Vos, G. Decision and detection limits for linear calibration curves. Anal. Chem. 42, 849–855 (1970).

    Article  CAS  Google Scholar 

  104. Lundquist, P.M. et al. Parallel confocal detection of single molecules in real time. Opt. Lett. 33, 1026–1028 (2008).

    Article  PubMed  Google Scholar 

  105. Bashford, G. et al. Automated bead-trapping apparatus and control system for single-molecule DNA sequencing. Opt. Express 16, 3445–3455 (2008).

    Article  CAS  PubMed  Google Scholar 

  106. Gibson, J.D. Principles of Digital and Analog Communications, edn. 2 (Macmillan Publishing, New York, 1993).

    Google Scholar 

  107. Honigs, D. The sayings of Tomas Hirschfeld. Applied Spect. 40, 11A (1986).

    Google Scholar 

  108. Hirschfeld, T. Limits of analysis. Anal. Chem. 48, 16A–31A (1976).

    Article  CAS  Google Scholar 

  109. Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002).

  110. Eckmann, J.-P. Trading codes for errors. Proc. Natl. Acad. Sci. USA 105, 8165–8166 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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