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The development and impact of 454 sequencing

Abstract

The 454 Sequencer has dramatically increased the volume of sequencing conducted by the scientific community and expanded the range of problems that can be addressed by the direct readouts of DNA sequence. Key breakthroughs in the development of the 454 sequencing platform included higher throughput, simplified all in vitro sample preparation and the miniaturization of sequencing chemistries, enabling massively parallel sequencing reactions to be carried out at a scale and cost not previously possible. Together with other recently released next-generation technologies, the 454 platform has started to democratize sequencing, providing individual laboratories with access to capacities that rival those previously found only at a handful of large sequencing centers. Over the past 18 months, 454 sequencing has led to a better understanding of the structure of the human genome, allowed the first non-Sanger sequence of an individual human and opened up new approaches to identify small RNAs. To make next-generation technologies more widely accessible, they must become easier to use and less costly. In the longer term, the principles established by 454 sequencing might reduce cost further, potentially enabling personalized genomics.

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Figure 1: Diagram of the pyrosequencing process.
Figure 2: Overview of the 454 sequencing technology.
Figure 3: Next-generation market segmentation.

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References

  1. 1

    Gilbert, W. & Maxam, A. The nucleotide sequence of the lac operator. Proc. Natl. Acad. Sci. USA 70, 3581–3584 (1973).

    CAS  Article  PubMed  Google Scholar 

  2. 2

    Sanger, F. The Croonian Lecture, 1975. Nucleotide sequences in DNA. Proc. R. Soc. Lond. B Biol. Sci. 191, 317–333 (1975).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Sanger, F. & Coulson, A.R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94, 441–448 (1975).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Khrapko, K.R. et al. An oligonucleotide hybridization approach to DNA sequencing. FEBS Lett. 256, 118–122 (1989).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Hansma, H.G. et al. Progress in sequencing deoxyribonucleic acid with an atomic force microscope. J. Vac. Sci. Technol. B 9, 1282–1284 (1991).

    CAS  Article  Google Scholar 

  6. 6

    Koster, H. et al. A strategy for rapid and efficient DNA sequencing by mass spectrometry. Nat. Biotechnol. 14, 1123–1128 (1996).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Hyman, E.D. A new method of sequencing DNA. Anal. Biochem. 174, 423–436 (1988).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Koster, H. et al. Oligonucleotide synthesis and multiplex DNA sequencing using chemiluminescent detection. Nucleic Acids Symp. Ser. 24 318–321 (1991).

    CAS  Google Scholar 

  9. 9

    Nyren, P., Pettersson, B. & Uhlen, M. Solid phase DNA minisequencing by an enzymatic luminometric inorganic pyrophosphate detection assay. Anal. Biochem. 208, 171–175 (1993).

    CAS  Article  PubMed  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Melamede, R.J. Automatable process for sequencing nucleotide. US patent 4,863, 849 (1985).

  12. 12

    Woolley, A.T. & Mathies, R.A. Ultra-high-speed DNA sequencing using capillary electrophoresis chips. Anal. Chem. 67, 3676–3680 (1995).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Cantor, C.R. & Smith, C. Genomics: The Science and Technology Behind The Human Genome Project, edn. 1 (Wiley-Interscience, Hoboken, NJ, 1999).

    Book  Google Scholar 

  14. 14

    Meldrum, D. Automation for genomics, part one: preparation for sequencing. Genome Res. 10, 1081–1092 (2000).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Christensen, C.M. The innovator's dilemma: when new technologies cause great firms to fail (Harvard Business School Press, Boston, 1997).

  16. 16

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Wheeler, D. et al. The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872–877 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Cox-Foster, D.L. et al. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318, 283–287 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Korbel, J.O. et al. Paired-end mapping reveals extensive structural variation in the human genome. Science 318, 420–426 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Palacios, G. et al. A new arenavirus in a cluster of fatal transplant-associated diseases. NEJM 358, 991–998 (2008).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Briggs, A.W. et al. Patterns of damage in genomic DNA sequences from a Neandertal. Proc. Natl. Acad. Sci. USA 104, 14616–14621 (2007).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Green, R.E. et al. Analysis of one million base pairs of Neanderthal DNA. Nature 444, 330–336 (2006).

    CAS  Article  PubMed  Google Scholar 

  23. 23

    Noonan, J.P. et al. Sequencing and analysis of Neanderthal genomic DNA. Science 314, 1113–1118 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Moore, G.E. Cramming more components onto integrated circuits. Electronics 38, 114–117 (1965).

    Google Scholar 

  25. 25

    Rusk, N. & Kiermer, V. Primer: sequencing—the next generation. Nat. Methods 5, 15 (2008).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. & Nyren, P. Real-time DNA sequencing using detection of pyrophosphate release. Anal. Biochem. 242, 84–89 (1996).

    CAS  Article  Google Scholar 

  27. 27

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

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Ronaghi, M. Pyrosequencing for SNP genotyping. Methods Mol. Biol. 212, 189–195 (2003).

    CAS  PubMed  Google Scholar 

  29. 29

    Fakhrai-Rad, H., Pourmand, N. & Ronaghi, M. Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms. Hum. Mutat. 19, 479–485 (2002).

    CAS  Article  PubMed  Google Scholar 

  30. 30

    Ronaghi, M. Pyrosequencing sheds light on DNA sequencing. Genome Res. 11, 3–11 (2001).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Steemers, F.J. & Walt, D.R. Multi-analyte sensing: from site-selective deposition to randomly-ordered addressable optical fiber sensors. Mikrochim. Acta 131, 99–105 (1999).

    CAS  Article  Google Scholar 

  32. 32

    Pantano, P. & Walt, D.R. Ordered nanowell arrays. Chem. Mater. 8, 2832–2835 (1996).

    CAS  Article  Google Scholar 

  33. 33

    Ferguson, J.A., Steemers, F.J. & Walt, D.R. High-density fiber-optic DNA random microsphere array. Anal. Chem. 72, 5618–5624 (2000).

    CAS  Article  PubMed  Google Scholar 

  34. 34

    Leamon, J.H. & Rothberg, J.M. Cramming more sequencing reactions onto microreactor chips. Chem. Rev. 107, 3367–3376 (2007).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Chen, Y.J. et al. Double ended sequencing. US patent 7,244,567. (2007).

  36. 36

    Tawfik, D.S. & Griffiths, A.D. Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652–656 (1998).

    CAS  Article  PubMed  Google Scholar 

  37. 37

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

    CAS  Article  Google Scholar 

  38. 38

    Nakano, M. et al. Single-molecule PCR using water-in-oil emulsion. J. Biotechnol. 102, 117–124 (2003).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Leamon, J.H. et al. A massively parallel PicoTiterPlate based platform for discrete picoliter-scale polymerase chain reactions. Electrophoresis 24, 3769–3777 (2003).

    CAS  Article  PubMed  Google Scholar 

  40. 40

    Edwards, R.A. et al. Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7, 57 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Fraser, C.M. et al. The minimal gene complement of Mycoplasma genitalium. Science 270, 397–403 (1995).

    CAS  PubMed  Google Scholar 

  42. 42

    Bender, W., Spierer, P. & Hogness, D.S. Chromosomal walking and jumping to isolate DNA from the Ace and rosy loci and the bithorax complex in Drosophila melanogaster . J. Mol. Biol. 168, 17–33 (1983).

    CAS  Article  PubMed  Google Scholar 

  43. 43

    Spierer, P., Spierer, A., Bender, W. & Hogness, D.S. Molecular mapping of genetic and chromomeric units in Drosophila melanogaster . J. Mol. Biol. 168, 35–50 (1983).

    CAS  Article  PubMed  Google Scholar 

  44. 44

    Andries, K. et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis . Science 307, 223–227 (2005).

    CAS  Article  PubMed  Google Scholar 

  45. 45

    Hiller, N.L. et al. comparative genomic analyses of seventeen Streptococcus pneumoniae strains: insights into the pneumococcal supragenome. J. Bacteriol. 189, 8186–8195 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Hofreuter, D. et al. Unique features of a highly pathogenic Campylobacter jejuni strain. Infect. Immun. 74, 4694–4707 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    Oh, J.D. et al. The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease progression. Proc. Natl. Acad. Sci. USA 103, 9999–10004 (2006).

    CAS  Article  PubMed  Google Scholar 

  48. 48

    Raymond, J.A., Fritsen, C. & Shen, K. An ice-binding protein from an Antarctic sea ice bacterium. FEMS Microbiol. Ecol. 61, 214–221 (2007).

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Smith, M.G. et al. New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. Genes Dev. 21, 601–614 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50

    Bainbridge, M.N. et al. Analysis of the prostate cancer cell line LNCaP transcriptome using a sequencing-by-synthesis approach. BMC Genomics 7, 246 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  51. 51

    Goldberg, S.M. et al. A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes. Proc. Natl. Acad. Sci. USA 103, 11240–11245 (2006).

    CAS  Article  PubMed  Google Scholar 

  52. 52

    Wicker, T. et al. 454 sequencing put to the test using the complex genome of barley. BMC Genomics 7, 275 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  53. 53

    Swaminathan, K., Varala, K. & Hudson, M.E. Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey. BMC Genomics 8, 132 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  54. 54

    Henderson, I.R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 38, 721–725 (2006).

    CAS  Article  PubMed  Google Scholar 

  55. 55

    Girard, A. l., Sachidanandam, R., Hannon, G.J. & Carmell, M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199–202 (2006).

    Article  PubMed  Google Scholar 

  56. 56

    Berezikov, E. et al. Diversity of microRNAs in human and chimpanzee brain. Nat. Genet. 38, 1375–1377 (2006).

    CAS  Article  PubMed  Google Scholar 

  57. 57

    Houwing, S. et al. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in zebrafish. Cell 129, 69–82 (2007).

    CAS  Article  PubMed  Google Scholar 

  58. 58

    Tarasov, V. et al. Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle 6, 1586–1593 (2007).

    CAS  Article  PubMed  Google Scholar 

  59. 59

    Weber, A.P.M., Weber, K.L., Carr, K., Wilkerson, C. & Ohlrogge, J.B. Sampling the Arabidopsis transcriptome with massively parallel pyrosequencing. Plant Physiol. 144, 32–42 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. 60

    Cheung, F. et al. Sequencing Medicago truncatula expressed sequenced tags using 454 Life Sciences technology. BMC Genomics 7, 272 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  61. 61

    Gowda, M. et al. Robust analysis of 5′-transcript ends (5′-RATE): a novel technique for transcriptome analysis and genome annotation. Nucleic Acids Res. 34, e126 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  62. 62

    Barbazuk, W.B., Emrich, S.J., Chen, H.D., Li, L. & Schnable, P.S. SNP discovery via 454 transcriptome sequencing. Plant J. 51, 910–918 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. 63

    Poinar, H.N. et al. Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science 311, 392–394 (2006).

    CAS  Article  PubMed  Google Scholar 

  64. 64

    Gilbert, M.T.P. et al. Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis. Nucleic Acids Res. 35, 1–10 (2007).

    CAS  Article  PubMed  Google Scholar 

  65. 65

    Stiller, M. et al. Inaugural article: patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA. Proc. Natl. Acad. Sci. USA 103, 13578–13584 (2006).

    CAS  Article  PubMed  Google Scholar 

  66. 66

    Sogin, M.L. et al. Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc. Natl. Acad. Sci. USA 103, 12115–12120 (2006).

    CAS  Article  PubMed  Google Scholar 

  67. 67

    Angly, F.E. et al. The marine viromes of four oceanic regions. PLoS Biol. 4, e368 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  68. 68

    Leininger, S. et al. Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442, 806–809 (2006).

    CAS  Article  Google Scholar 

  69. 69

    Krause, L. et al. Finding novel genes in bacterial communities isolated from the environment. Bioinformatics 22, e281–e289 (2006).

    CAS  Article  PubMed  Google Scholar 

  70. 70

    Pennisi, E. Breakthrough of the year: human genetic variation. Science 318, 1842–1843 (2007).

    CAS  Article  PubMed  Google Scholar 

  71. 71

    Steemers, F.J., Ferguson, J.A. & Walt, D.R. Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays. Nat. Biotechnol. 18, 91–94 (2000).

    CAS  Article  PubMed  Google Scholar 

  72. 72

    Levy, S. et al. The diploid genome sequence of an individual human. PLoS Biol. 5, e254 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  73. 73

    Hayden, E. Personalized genomes go mainstream. Nature (News) 250, 11 (2007).

    Article  Google Scholar 

  74. 74

    Karow, J. Next-gen sequencers improve in '07; vendors promise more gains in 2008. In Sequence 2 (2008).

    Google Scholar 

  75. 75

    Egholm, M. Why length matters in next generation sequencing. Presented at Cambridge Healthtech Institute's Exploring Next Generation Sequencing meeting, Providence, RI. October 17–18, 2007.

  76. 76

    Mitra, R.D. & Church, G.M. In situ localized amplification and contact replication of many individual DNA molecules. Nucleic Acids Res. 27, e34 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  77. 77

    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).

    CAS  Article  Google Scholar 

  78. 78

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

    CAS  Article  Google Scholar 

  79. 79

    Braslavsky, I., Hebert, B., Kartalov, E. & Quake, S.R. Sequence information can be obtained from single DNA molecules. Proc. Natl. Acad. Sci. USA 100, 3960–3964 (2003).

    CAS  Article  Google Scholar 

  80. 80

    Efcavitch, W. The HeliScope single molecule sequencer: an integrated genetic analyzer for true single molecule sequencing. Presented at Advances in Genome Biology and Technology meeting, Marco Island, FL, February 7–9, 2008.

  81. 81

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

    CAS  Article  Google Scholar 

  82. 82

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

    CAS  Article  Google Scholar 

  83. 83

    Turner, S. Harnessing nature's powerful DNA sequencing engine: single molecule real time sequencing-by-synthesis. Presented at Advances in Genome Biology and Technology meeting, Marco Island, FL, February 7–9, 2008.

  84. 84

    Illumina. Illumina sequences the first African human genome (Illumina, San Diego) http://investor.illumina.com/phoenix.zhtml?c=121127&p=irol-newsArticle&ID=1105194&highlight (6 February 2008)

  85. 85

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

    CAS  Article  Google Scholar 

  86. 86

    Campbell, P.J. et al. Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing. Nat. Genet. 40, 722–729 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Correspondence to Jonathan M Rothberg.

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The authors have financial interests in a number of companies operating in fields that directly benefit from the promotion and success of next-generation sequencing technology in general. Financial interest includes stock ownership, stock options, salaries, consulting fees and bonuses. Currently these financial interests are in RainDance Technologies, and ION Torrent systems. In the past the authors were also key employees, and/or founders, of 454 Life Sciences, are named inventors on key 454 patents, and benefitted from the commercial, scientific and financial success of 454 Life Sciences.

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Rothberg, J., Leamon, J. The development and impact of 454 sequencing. Nat Biotechnol 26, 1117–1124 (2008). https://doi.org/10.1038/nbt1485

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