Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Next-generation DNA sequencing

Abstract

DNA sequence represents a single format onto which a broad range of biological phenomena can be projected for high-throughput data collection. Over the past three years, massively parallel DNA sequencing platforms have become widely available, reducing the cost of DNA sequencing by over two orders of magnitude, and democratizing the field by putting the sequencing capacity of a major genome center in the hands of individual investigators. These new technologies are rapidly evolving, and near-term challenges include the development of robust protocols for generating sequencing libraries, building effective new approaches to data-analysis, and often a rethinking of experimental design. Next-generation DNA sequencing has the potential to dramatically accelerate biological and biomedical research, by enabling the comprehensive analysis of genomes, transcriptomes and interactomes to become inexpensive, routine and widespread, rather than requiring significant production-scale efforts.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Work flow of conventional versus second-generation sequencing.
Figure 2: Clonal amplification of sequencing features.
Figure 3: Strategies for cyclic array sequencing.

Similar content being viewed by others

References

  1. Hutchison, C.A., III. DNA sequencing: bench to bedside and beyond. Nucleic Acids Res. 35, 6227–6237 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sanger, F. Sequences, sequences, and sequences. Annu. Rev. Biochem. 57, 1–28 (1988).

    Article  CAS  PubMed  Google Scholar 

  3. Sanger, F. et al. Nucleotide sequence of bacteriophage phi X174 DNA. Nature 265, 687–695 (1977).

    Article  CAS  PubMed  Google Scholar 

  4. Shendure, J., Mitra, R.D., Varma, C. & Church, G.M. Advanced sequencing technologies: methods and goals. Nat. Rev. Genet. 5, 335–344 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Swerdlow, H., Wu, S.L., Harke, H. & Dovichi, N.J. Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette. J. Chromatogr. 516, 61–67 (1990).

    Article  CAS  PubMed  Google Scholar 

  6. Hunkapiller, T., Kaiser, R.J., Koop, B.F. & Hood, L. Large-scale and automated DNA sequence determination. Science 254, 59–67 (1991).

    Article  CAS  PubMed  Google Scholar 

  7. Ewing, B. & Green, P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8, 186–194 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Ewing, B., Hillier, L., Wendl, M.C. & Green, P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. Blazej, R.G., Kumaresan, P. & Mathies, R.A. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. Proc. Natl. Acad. Sci. USA 103, 7240–7245 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gresham, D., Dunham, M.J. & Botstein, D. Comparing whole genomes using DNA microarrays. Nat. Rev. Genet. 9, 291–302 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Soni, G.V. & Meller, A. Progress toward ultrafast DNA sequencing using solid-state nanopores. Clin. Chem. 53, 1996–2001 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Healy, K. Nanopore-based single-molecule DNA analysis. Nanomed. 2, 459–481 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Mitra, R.D., Shendure, J., Olejnik, J., Edyta Krzymanska, O. & Church, G.M. Fluorescent in situ sequencing on polymerase colonies. Anal. Biochem. 320, 55–65 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Bentley, D.R. Whole-genome re-sequencing. Curr. Opin. Genet. Dev. 16, 545–552 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Ng, P. et al. Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation. Nat. Methods 2, 105–111 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. 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 

  21. Adessi, C. et al. Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms. Nucleic Acids Res. 28, e87 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fedurco, M., Romieu, A., Williams, S., Lawrence, I. & Turcatti, G. BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies. Nucleic Acids Res. 34, e22 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. 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).

    Article  CAS  PubMed  Google Scholar 

  26. McKernan, K., Blanchard, A., Kotler, L. & Costa, G. Reagents, methods, and libraries for bead-based sequencing. US patent application 20080003571 (2006).

  27. Housby, J.N. & Southern, E.M. Fidelity of DNA ligation: a novel experimental approach based on the polymerisation of libraries of oligonucleotides. Nucleic Acids Res. 26, 4259–4266 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Macevicz, S.C. DNA sequencing by parallel oligonucleotide extensions. US patent 5750341 (1998).

  29. 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 

  30. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sjoblom, T. et al. The consensus coding sequences of human breast and colorectal cancers. Science 314, 268–274 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Wood, L.D. et al. The genomic landscapes of human breast and colorectal cancers. Science 318, 1108–1113 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Brockman, W. et al. Quality scores and SNP detection in sequencing-by-synthesis systems. Genome Res. 18, 763–770 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Quinlan, A.R., Stewart, D.A., Stromberg, M.P. & Marth, G.T. Pyrobayes: an improved base caller for SNP discovery in pyrosequences. Nat. Methods 5, 179–181 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Li, R., Li, Y., Kristiansen, K. & Wang, J. SOAP: short oligonucleotide alignment program. Bioinformatics 24, 713–714 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Ning, Z., Cox, A.J. & Mullikin, J.C. SSAHA: a fast search method for large DNA databases. Genome Res. 11, 1725–1729 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. published online, doi:10.1101/gr.078212.108 (19 August 2008).

  38. Butler, J. et al. ALLPATHS: de novo assembly of whole-genome shotgun microreads. Genome Res. 18, 810–820 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sundquist, A., Ronaghi, M., Tang, H., Pevzner, P. & Batzoglou, S. Whole-genome sequencing and assembly with high-throughput, short-read technologies. PLoS ONE 2, e484 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Warren, R.L., Sutton, G.G., Jones, S.J. & Holt, R.A. Assembling millions of short DNA sequences using SSAKE. Bioinformatics 23, 500–501 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Zerbino, D.R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Brazma, A. et al. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat. Genet. 29, 365–371 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. Wheeler, D.L. et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 36, D13–D21 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Wheeler, D.A. et al. The complete genome of an individual by massively parallel DNA sequencing. Nature 452, 872–876 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Van Tassell, C.P. et al. SNP discovery and allele frequency estimation by deep sequencing of reduced representation libraries. Nat. Methods 5, 247–252 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Dahl, F. et al. Multigene amplification and massively parallel sequencing for cancer mutation discovery. Proc. Natl. Acad. Sci. USA 104, 9387–9392 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Fredriksson, S. et al. Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector. Nucleic Acids Res. 35, e47 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  49. Bashiardes, S. et al. Direct genomic selection. Nat. Methods 2, 63–69 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. 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 

  51. Hodges, E. et al. Genome-wide in situ exon capture for selective resequencing. Nat. Genet. 39, 1522–1527 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Okou, D.T. et al. Microarray-based genomic selection for high-throughput resequencing. Nat. Methods 4, 907–909 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Chen, W. et al. Mapping translocation breakpoints by next-generation sequencing. Genome Res. 18, 1143–1149 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  56. Wilhelm, B.T. et al. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature 453, 1239–1243 (2008).

    Article  CAS  PubMed  Google Scholar 

  57. Sugarbaker, D.J. et al. Transcriptome sequencing of malignant pleural mesothelioma tumors. Proc. Natl. Acad. Sci. USA 105, 3521–3526 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  PubMed  Google Scholar 

  60. Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis . Cell 133, 523–536 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 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 

  62. Cloonan, N. et al. Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat. Methods 5, 613–619 (2008).

    Article  CAS  PubMed  Google Scholar 

  63. 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  CAS  PubMed  PubMed Central  Google Scholar 

  64. Morin, R.D. et al. Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res. 18, 610–621 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Korshunova, Y. et al. Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA. Genome Res. 18, 19–29 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Ordway, J.M. et al. Identification of novel high-frequency DNA methylation changes in breast cancer. PLoS ONE 2, e1314 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B. Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).

    Article  CAS  PubMed  Google Scholar 

  68. Robertson, G. et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat. Methods 4, 651–657 (2007).

    Article  CAS  PubMed  Google Scholar 

  69. Schones, D.E. et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887–898 (2008).

    Article  CAS  PubMed  Google Scholar 

  70. Wold, B. & Myers, R.M. Sequence census methods for functional genomics. Nat. Methods 5, 19–21 (2008).

    Article  CAS  PubMed  Google Scholar 

  71. Meyer, M., Stenzel, U. & Hofreiter, M. Parallel tagged sequencing on the 454 platform. Nat. Protocols 3, 267–278 (2008).

    Article  CAS  PubMed  Google Scholar 

  72. Slater, G.S. & Birney, E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6, 31 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Smith, A.D., Xuan, Z. & Zhang, M.Q. Using quality scores and longer reads improves accuracy of Solexa read mapping. BMC Bioinformatics 9, 128 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Hernandez, D., Francois, P., Farinelli, L., Osteras, M. & Schrenzel, J. De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer. Genome Res. 18, 802–809 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Chaisson, M.J. & Pevzner, P.A. Short read fragment assembly of bacterial genomes. Genome Res. 18, 324–330 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Dohm, J.C., Lottaz, C., Borodina, T. & Himmelbauer, H. SHARCGS, a fast and highly accurate short-read assembly algorithm for de novo genomic sequencing. Genome Res. 17, 1697–1706 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Jeck, W.R. et al. Links extending assembly of short DNA sequences to handle error. Bioinformatics 23, 2942–2944 (2007).

    Article  CAS  PubMed  Google Scholar 

  78. Paegel, B.M., Blazej, R.G. & Mathies, R.A. Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis. Curr. Opin. Biotechnol. 14, 42–50 (2003).

    Article  CAS  PubMed  Google Scholar 

  79. Hong, J.W. & Quake, S.R. Integrated nanoliter systems. Nat. Biotechnol. 21, 1179–1183 (2003).

    Article  CAS  PubMed  Google Scholar 

  80. Emrich, C.A., Tian, H., Medintz, I.L. & Mathies, R.A. Microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis. Anal. Chem. 74, 5076–5083 (2002).

    Article  CAS  PubMed  Google Scholar 

  81. Toriello, N.M., Liu, C.N., Blazej, R.G., Thaitrong, N. & Mathies, R.A. Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis. Anal. Chem. 79, 8549–8556 (2007).

    Article  CAS  PubMed  Google Scholar 

  82. Blazej, R.G., Kumaresan, P., Cronier, S.A. & Mathies, R.A. Inline injection microdevice for attomole-scale sanger DNA sequencing. Anal. Chem. 79, 4499–4506 (2007).

    Article  CAS  PubMed  Google Scholar 

  83. Hong, J.W., Studer, V., Hang, G., Anderson, W.F. & Quake, S.R. A nanoliter-scale nucleic acid processor with parallel architecture. Nat. Biotechnol. 22, 435–439 (2004).

    Article  CAS  PubMed  Google Scholar 

  84. Patil, N. et al. Blocks of limited haplotype diversity revealed by high-resolution scanning of human chromosome 21. Science 294, 1719–1723 (2001).

    Article  CAS  PubMed  Google Scholar 

  85. Frazer, K.A. et al. A sequence-based variation map of 8.27 million SNPs in inbred mouse strains. Nature 448, 1050–1053 (2007).

    Article  CAS  PubMed  Google Scholar 

  86. Gresham, D. et al. Genome-wide detection of polymorphisms at nucleotide resolution with a single DNA microarray. Science 311, 1932–1936 (2006).

    Article  CAS  PubMed  Google Scholar 

  87. Albert, T.J. et al. Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori. Nat. Methods 2, 951–953 (2005).

    Article  CAS  PubMed  Google Scholar 

  88. Drmanac, S. et al. Accurate sequencing by hybridization for DNA diagnostics and individual genomics. Nat. Biotechnol. 16, 54–58 (1998).

    Article  CAS  PubMed  Google Scholar 

  89. Drmanac, R., Labat, I., Brukner, I. & Crkvenjakov, R. Sequencing of megabase plus DNA by hybridization: theory of the method. Genomics 4, 114–128 (1989).

    Article  CAS  PubMed  Google Scholar 

  90. 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 

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

    Article  CAS  PubMed  Google Scholar 

  92. Deamer, D.W. & Akeson, M. Nanopores and nucleic acids: prospects for ultrarapid sequencing. Trends Biotechnol. 18, 147–151 (2000).

    Article  CAS  PubMed  Google Scholar 

  93. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. & Branton, D. Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl. Acad. Sci. USA 97, 1079–1084 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Cockroft, S.L., Chu, J., Amorin, M. & Ghadiri, M.R. A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution. J. Am. Chem. Soc. 130, 818–820 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. 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 

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

    Article  PubMed  Google Scholar 

  97. 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 

Download references

Acknowledgements

We thank G. Church and G. Porreca for helpful comments on the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Jay Shendure or Hanlee Ji.

Ethics declarations

Competing interests

J.S. has patents pending in the field of next-generation sequencing technology.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shendure, J., Ji, H. Next-generation DNA sequencing. Nat Biotechnol 26, 1135–1145 (2008). https://doi.org/10.1038/nbt1486

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1486

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing