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.

Coming of age: ten years of next-generation sequencing technologies

Key Points

  • There are two major paradigms in next-generation sequencing (NGS) technology: short-read sequencing and long-read sequencing. Short-read sequencing approaches provide lower-cost, higher-accuracy data that are useful for population-level research and clinical variant discovery. By contrast, long-read approaches provide read lengths that are well suited for de novo genome assembly applications and full-length isoform sequencing.

  • NGS technologies have been evolving over the past 10 years, leading to substantial improvements in quality and yield; however, certain approaches have proven to be more effective and adaptable than others.

  • Recent improvements in chemistry, costs, throughput and accessibility are driving the emergence of new, varied technologies to address applications that were not previously possible. These include integrated long-read and short-read sequencing studies, routine clinical DNA sequencing, real-time pathogen DNA monitoring and massive population-level projects.

  • Although massive strides are being made in this technology, several notable limitations remain. The time required to sequence and analyse data limits the use of NGS in clinical applications in which time is an important factor; the costs and error rates of long-read sequencing make it prohibitive for routine use, and ethical considerations can limit the public and private use of genetic data.

  • We can expect increasing democratization and options for NGS in the future. Many new instruments with varied chemistries and applications are being released or being developed.

Abstract

Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Template amplification strategies.
Figure 2: Sequencing by ligation methods.
Figure 3: Sequencing by synthesis: cyclic reversible termination approaches.
Figure 4: Sequencing by synthesis: single-nucleotide addition approaches.
Figure 5: Real-time and synthetic long-read sequencing approaches.

References

  1. Watson, J. D. & Crick, F. H. The structure of DNA. Cold Spring Harb. Symp. Quant. Biol. 18, 123–131 (1953).

    Article  CAS  PubMed  Google Scholar 

  2. Mardis, E. R. Next-generation sequencing platforms. Annu. Rev. Anal. Chem. (Palo Alto Calif.) 6, 287–303 (2013). This article provides a concise description of technological advancements supporting NGS.

    Article  CAS  Google Scholar 

  3. Wetterstrand, K. A. DNA sequencing costs: data from the NHGRI Genome Sequencing Program (GSP). National Human Genome Research Institute [online], http://www.genome.gov/sequencingcosts (updated 15 Jan 2016).

  4. Kircher, M. & Kelso, J. High-throughput DNA sequencing — concepts and limitations. Bioessays 32, 524–536 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Veritas Genetics. Veritas genetics launches $999 whole genome and sets new standard for genetic testing — Press Release. Veritas Genetics [online], https://www.veritasgenetics.com/documents/VG-launches-999-whole-genome.pdf (updated 4 Mar 2016).

  6. Veritas Genetics. Veritas genetics breaks $1,000 whole genome barrier — Press Release. Veritas Genetics [online], https://www.veritasgenetics.com/documents/VG-PGP-Announcement-Final.pdf (29 Sep 2015).

  7. Liu, L. et al. Comparison of next-generation sequencing systems. J. Biomed. Biotechnol. 2012, 251364 (2012).

    PubMed  PubMed Central  Google Scholar 

  8. 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 

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

    Article  CAS  PubMed  Google Scholar 

  10. 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 

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

    Article  CAS  PubMed  Google Scholar 

  12. 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  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Drmanac, R. et al. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 327, 78–81 (2010). This paper describes the use of DNA nanoballs to achieve clonal amplification and the use of cPAL to achieve human genome sequencing as implemented by Complete Genomics (BGI).

    Article  CAS  PubMed  Google Scholar 

  15. Tomkinson, A. E., Vijayakumar, S., Pascal, J. M. & Ellenberger, T. DNA ligases: structure, reaction mechanism, and function. Chem. Rev. 106, 687–699 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Landegren, U., Kaiser, R., Sanders, J. & Hood, L. A ligase-mediated gene detection technique. Science 241, 1077–1080 (1988).

    Article  CAS  PubMed  Google Scholar 

  17. Valouev, A. et al. A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning. Genome Res. 18, 1051–1063 (2008). This paper describes the use of cleavable two-base-encoded probes to achieve genome-wide nucleosome mapping in Caenorhabditis elegans . This technology is implemented by Applied Biosystems (Thermo Fisher) for the SOLiD platform.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Metzker, M. L. Sequencing technologies — the next generation. Nat. Rev. Genet. 11, 31–46 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. 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 

  20. 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 

  21. Timmerman, L. DNA sequencing market will exceed $20 billion, says Illumina CEO Jay Flatley. Forbes [online], http://www.forbes.com/sites/luketimmerman/2015/04/29/qa-with-jay-flatley-ceo-of-illumina-the-genomics-company-pursuing-a-20b-market/#4dbd19943bf5 (29 Apr 2015).

  22. Karow, J. Qiagen launches GeneReader NGS System at AMP; presents performance evaluation by broad. GenomeWeb [online], https://www.genomeweb.com/molecular-diagnostics/qiagen-launches-genereader-ngs-system-amp-presents-performance-evaluation (4 Nov 2015).

  23. Smith, D. R. & McKernan, K. Methods of producing and sequencing modified polynucleotides. US Patent 8058030 (2011).

  24. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005). This paper describes the development of the first NGS technology through the use of pyrosequencing. The authors demonstrate this method through sequencing of the Mycoplasma genitalium genome.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rothberg, J. M. et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature 475, 348–352 (2011). This paper describes the first non-optical sequencing technology using a massively parallel semi-conductor device to monitor H+ release during DNA synthesis, as implemented by the Ion Torrent platform (Thermo Fisher). The authors demonstrate this technology by sequencing both bacterial and human DNA.

    Article  CAS  PubMed  Google Scholar 

  26. Rieber, N. et al. Coverage bias and sensitivity of variant calling for four whole-genome sequencing technologies. PLoS ONE 8, e66621 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zook, J. M. et al. Integrating human sequence data sets provides a resource of benchmark SNP and indel genotype calls. Nat. Biotechnol. 32, 246–251 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Nothnagel, M. et al. Technology-specific error signatures in the 1000 Genomes Project data. Hum. Genet. 130, 505–516 (2011).

    Article  PubMed  Google Scholar 

  29. Shen, Y. Sarin, S., Liu, Y., Hobert, O. & Pe'er, I. Comparing platforms for C. elegans mutant identification using high-throughput whole-genome sequencing. PLoS ONE 3, e4012 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Chan, M. et al. Development of a next-generation sequencing method for BRCA mutation screening: a comparison between a high-throughput and a benchtop platform. J. Mol. Diagnost. 14, 602–612 (2012).

    Article  CAS  Google Scholar 

  31. Wall, J. D. et al. Estimating genotype error rates from high-coverage next-generation sequence data. Genome Res. 24, 1734–1739 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Harismendy, O. et al. Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome Biol. 10, R32 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. BGI. Revolocity Whole Genome Sequencing Service — Press Release. BGI [online], http://u70g92ptbyk941g21dd41fc4.wpengine.netdna-cdn.com/wp-content/uploads/2015/10/Global-WGSRevolocity-ENG-10-15.pdf (2015).

  34. Karow, J. BGI halts revolocity launch, cuts complete genomics staff as part of strategic shift. GenomeWeb [online], https://www.genomeweb.com/sequencing-technology/bgi-halts-revolocity-launch-cuts-complete-genomics-staff-part-strategic-shift (23 Nov 2015).

  35. Bentley, D. R. et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456, 53–59 (2008). This paper demonstrates the use of reversible dye-terminator chemistry for human genome sequencing. This platform is used by the Illumina suite of platforms.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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 

  37. Nakamura, K. et al. Sequence-specific error profile of Illumina sequencers. Nucleic Acids Res. 39, e90 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Minoche, A. E., Dohm, J. C. & Himmelbauer, H. Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and genome analyzer systems. Genome Biol. 12, R112 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ley, T. J. et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 456, 66–72 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sarin, S., Prabhu, S., O'Meara, M. M., Pe'er, I. & Hobert, O. Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nat. Methods 5, 865–867 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Park, P. J. ChIP–seq: advantages and challenges of a maturing technology. Nat. Rev. Genet. 10, 669–680 (2009). This review provides an overview of ChIP–seq methods for detecting chromatin–DNA interactions and their importance to epigenetics research.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Brunner, A. L. et al. Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. Genome Res. 19, 1044–1056 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57–63 (2009). This review provides an overview of advances and challenges in techniques that are used in transcriptomic research with a specific focus in methods that use NGS technologies.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang, X. et al. A trimming-and-retrieving alignment scheme for reduced representation bisulfite sequencing. Bioinformatics 31, 2040–2042 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Qiagen. Oncology insights enabled by knowledge base-guided panel design and the seamless workflow of the GeneReader NGS system — Press Release. Qiagen [online], http://www.genereaderngs.com/PROM-9192-001_1100403_WP_GeneReader_NGS_0116_NA.pdf (2016).

  47. Forgetta, V. et al. Sequencing of the Dutch elm disease fungus genome using the Roche/454 GS-FLX Titanium System in a comparison of multiple genomics core facilities. J. Biomol. Tech. 24, 39–49 (2013).

    PubMed  PubMed Central  Google Scholar 

  48. Loman, N. J. et al. Performance comparison of benchtop high-throughput sequencing platforms. Nat. Biotechnol. 30, 434–439 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. GenomeWeb. Roche shutting down 454 sequencing business. GenomeWeb [online], https://www.genomeweb.com/sequencing/roche-shutting-down-454-sequencing-business (15 Oct 2015).

  50. Malapelle, U. et al. Ion Torrent next-generation sequencing for routine identification of clinically relevant mutations in colorectal cancer patients. J. Clin. Pathol. 68, 64–68 (2015).

    Article  PubMed  Google Scholar 

  51. Li, S. et al. Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study. Nat. Biotechnol. 32, 915–925 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Life Technologies. Ion semiconductor sequencing uniquely enables both accurate long reads and paired-end sequencing. Life Technologies [online], https://www3.appliedbiosystems.com/cms/groups/applied_markets_marketing/documents/generaldocuments/cms_098680.pdf (2011)

  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. McCarroll, S. A. & Altshuler, D. M. Copy-number variation and association studies of human disease. Nat. Genet. 39, S37–S42 (2007).

    Article  CAS  PubMed  Google Scholar 

  55. Mirkin, S. M. Expandable DNA repeats and human disease. Nature 447, 932–940 (2007).

    Article  CAS  PubMed  Google Scholar 

  56. Stankiewicz, P. & Lupski, J. R. Structural variation in the human genome and its role in disease. Annu. Rev. Med. 61, 437–455 (2010).

    Article  CAS  PubMed  Google Scholar 

  57. Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009). The authors describe the development of a real-time sequencing method using their zero-mode waveguide sensors as implemented by the Pacific Biosciences platform. The authors demonstrate the technique by sequencing synthetic DNA templates.

    Article  CAS  PubMed  Google Scholar 

  58. 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 

  59. Loomis, E. W. et al. Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. Genome Res. 23, 121–128 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing. Nat. Nanotechnol. 4, 265–270 (2009). The authors demonstrate the use of a mutant alpha-hemolysin for ordered, continuous detection of free nucleotides in solution. This work provides the basis for the approach used by ONT.

    Article  CAS  PubMed  Google Scholar 

  61. Voskoboynik, A. et al. The genome sequence of the colonial chordate, Botryllus schlosseri. eLife 2, e00569 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  62. McCoy, R. C. et al. Illumina TruSeq synthetic long-reads empower de novo assembly and resolve complex, highly-repetitive transposable elements. PLoS ONE 9, e106689 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Schatz, M. C., Delcher, A. L. & Salzberg, S. L. Assembly of large genomes using second-generation sequencing. Genome Res. 20, 1165–1173 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. English, A. C. et al. Assessing structural variation in a personal genome-towards a human reference diploid genome. BMC Genomics 16, 286 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Carneiro, M. O. et al. Pacific Biosciences sequencing technology for genotyping and variation discovery in human data. BMC Genomics 13, 375 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Quail, M. A. et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13, 341 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Koren, S. et al. Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat. Biotechnol. 30, 693–700 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Larsen, P. A., Heilman, A. M. & Yoder, A. D. The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms. BMC Genomics 15, 720 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  69. Heger, M. PacBio launches higher-throughput, lower-cost single-molecule sequencing system. GenomeWeb [online], https://www.genomeweb.com/business-news/pacbio-launches-higher-throughput-lower-cost-single-molecule-sequencing-system (01 Oct 2015).

  70. Goodwin, S. et al. Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome. Genome Res. 25, 1750–1756 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Jain, M. et al. Improved data analysis for the MinION nanopore sequencer. Nat. Methods 12, 351–356 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Heger, M. 10X Genomics, Pacific Biosciences provide business updates at JP Morgan Healthcare Conference. GenomeWeb [online], https://www.genomeweb.com/sequencing-technology/10x-genomics-pacific-biosciences-provide-business-updates-jp-morgan-healthcare (13 Jan 2016).

  73. Cirulli, E. T. & Goldstein, D. B. Uncovering the roles of rare variants in common disease through whole-genome sequencing. Nat. Rev. Genet. 11, 415–425 (2010). This review provides a comprehensive overview of advances in, and challenges of using, WGS for variant discovery in human disease.

    Article  CAS  PubMed  Google Scholar 

  74. Ellis, M. J. et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature 486, 353–360 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Prat, A. & Perou, C. M. Mammary development meets cancer genomics. Nat. Med. 15, 842–844 (2009).

    Article  CAS  PubMed  Google Scholar 

  76. 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).

  77. 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015).

  78. Sudmant, P. H. et al. An integrated map of structural variation in 2,504 human genomes. Nature 526, 75–81 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. UK10K Consortium. The UK10K project identifies rare variants in health and disease. Nature 526, 82–90 (2015).

  80. Gudbjartsson, D. F. et al. Large-scale whole-genome sequencing of the Icelandic population. Nat. Genet. 47, 435–444 (2015).

    Article  CAS  PubMed  Google Scholar 

  81. Regalado, A. U.S. to develop DNA study of one million people. MIT Technology Review [online], http://www.technologyreview.com/news/534591/us-to-develop-dna-study-of-one-million-people (30 Jan 2015).

  82. Sidore, C. et al. Genome sequencing elucidates Sardinian genetic architecture and augments association analyses for lipid and blood inflammatory markers. Nat. Genet. 47, 1272–1281 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Hodges, E. et al. Genome-wide in situ exon capture for selective resequencing. Nat. Genet. 39, 1522–1527 (2015). This paper describes the in situ capture and selective enrichment of human exons for downstream NGS. This manuscript provides the methodological basis for whole-exome and targeted sequencing.

    Article  CAS  Google Scholar 

  84. Iossifov, I. et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature 515, 216–221 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. O'Roak, B. J. et al. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations. Nature 485, 246–250 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Sanders, S. J. et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature 485, 237–241 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Griffith, M. et al. Optimizing cancer genome sequencing and analysis. Cell Syst. 1, 210–223 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Rauch, C. et al. Towards an understanding of DNA recognition by the methyl-CpG binding domain 1. J. Biomol. Struct. Dyn. 22, 695–706 (2005).

    Article  CAS  PubMed  Google Scholar 

  89. Oda, M. et al. High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers. Nucleic Acids Res. 37, 3829–3839 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Irizarry, R. A. et al. Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Res. 18, 780–790 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33, 5868–5877 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Flusberg, B. A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat. Methods 7, 461–465 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Wescoe, Z. L., Schreiber, J. & Akeson, M. Nanopores discriminate among five C5-cytosine variants in DNA. J. Am. Chem. Soc. 136, 16582–16587 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Lam, E. T. et al. Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly. Nat. Biotechnol. 30, 771–776 (2012).

    Article  CAS  PubMed  Google Scholar 

  95. Eichler, E. E., Clark, R. A. & She, X. An assessment of the sequence gaps: unfinished business in a finished human genome. Nat. Rev. Genet. 5, 345–354 (2004).

    Article  CAS  PubMed  Google Scholar 

  96. Chaisson, M. J., Wilson, R. K. & Eichler, E. E. Genetic variation and the de novo assembly of human genomes. Nat. Rev. Genet. 16, 627–640 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Chaisson, M. J. et al. Resolving the complexity of the human genome using single-molecule sequencing. Nature 517, 608–611 (2015). This article provides strong support for the utility of long-read sequencing for generating high-quality reference genomes. The authors demonstrate this by closing and/or extending gaps and resolving structural variants in the GRCh37 human reference genome.

    Article  CAS  PubMed  Google Scholar 

  98. Ritz, A. et al. Characterization of structural variants with single molecule and hybrid sequencing approaches. Bioinformatics 30, 3458–3466 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Snyder, M. W., Adey, A., Kitzman, J. O. & Shendure, J. Haplotype-resolved genome sequencing: experimental methods and applications. Nat. Rev. Genet. 16, 344–358 (2015).

    Article  CAS  PubMed  Google Scholar 

  100. Kuleshov, V. et al. Whole-genome haplotyping using long reads and statistical methods. Nat. Biotechnol. 32, 261–266 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Treutlein, B. et al. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature 509, 371–375 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Sharon, D., Tilgner, H., Grubert, F. & Snyder, M. A single-molecule long-read survey of the human transcriptome. Nat. Biotechnol. 31, 1009–1014 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Quick, J. et al. Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella. Genome Biol. 16, 114 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  104. Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 530, 228–232 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. GenomeWeb. White House announces efforts to accelerate precision medicine initiative. GenomeWeb [online], https://www.genomeweb.com/molecular-diagnostics/white-house-announces-efforts-accelerate-precision-medicine-initiative (25 Feb 2016).

  106. Illumina. Illumina forms new company to enable early cancer detection via blood-based screening — Press Release. Illumina [online], http://www.illumina.com/company/news-center/press-releases/press-release-details.html?newsid=2127903 (10 Jan 2016).

  107. Schatz, M. C. & Langmead, B. The DNA data deluge: fast, efficient genome sequencing machines are spewing out more data than geneticists can analyze. IEEE Spectr. 50, 26–33 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  108. Pop, M. & Salzberg, S. L. Bioinformatics challenges of new sequencing technology. Trends Genet. 24, 142–149 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Sunyaev, S. R. Inferring causality and functional significance of human coding DNA variants. Hum. Mol. Genet. 21, R10–R17 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Gargis, A. S. et al. Assuring the quality of next-generation sequencing in clinical laboratory practice. Nat. Biotechnol. 30, 1033–1036 (2012).

    Article  CAS  PubMed  Google Scholar 

  111. Chrystoja, C. C. & Diamandis, E. P. Whole genome sequencing as a diagnostic test: challenges and opportunities. Clin. Chem. 60, 724–733 (2014).

    Article  CAS  PubMed  Google Scholar 

  112. McGuire, A. L. et al. Point-counterpoint. Ethics and genomic incidental findings. Science 340, 1047–1048 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Bowers, J. et al. Virtual terminator nucleotides for next-generation DNA sequencing. Nat. Methods 6, 593–595 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Heger, M. China's Direct Genomics unveils new targeted NGS system based on Helicos Tech for clinical use. GenomeWeb [online], https://www.genomeweb.com/business-news/chinas-direct-genomics-unveils-new-targeted-ngs-system-based-helicos-tech-clinical-use (27 Oct 2015).

  115. Karow, J. Oxford Nanopore presents details on new high-throughput sequencer, improvements to MinIon. GenomeWeb [online], https://www.genomeweb.com/sequencing/oxford-nanopore-presents-details-new-high-throughput-sequencer-improvements-mini (16 Sep 2014).

  116. Karow, J. Sigma-Aldrich enters co-marketing agreement with GenapSys for Genius sequencer. GenomeWeb [online], https://www.genomeweb.com/sequencing-technology/sigma-aldrich-enters-co-marketing-agreement-genapsys-genius-sequencer (1 Jul 2015).

  117. Roche. Roche acquires Genia Technologies to strengthen next generation sequencing pipeline — Press Release. Roche [online], http://www.roche.com/media/store/releases/med-cor-2014-06-02.htm (2 Jun 2014).

  118. Heger, M. Illumina unveils mini targeted sequencer, semiconductor sequencing project at JP Morgan Conference. GenomeWeb [online], https://www.genomeweb.com/sequencing-technology/illumina-unveils-mini-targeted-sequencer-semiconductor-sequencing-project-jp (1 Jan 2016).

  119. NanoString. NanoString Technologies presents proof-of-concept data for novel massively parallel single molecule sequencing chemistry at AGBT meeting — Press Release. NanoString [online], http://investors.nanostring.com/releasedetail.cfm?ReleaseID=954517 (11 Feb 2016).

  120. Raz, T. & Pascaline, M. Nucleic acid target detection using a detector, a probe and an inhibitor. US Patent 20130344485 (2013).

  121. Mankos, M. et al. A novel low energy electron microscope for DNA sequencing and surface analysis. Ultramicroscopy 145, 36–49 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Augenlicht, L. H. & Kobrin, D. Cloning and screening of sequences expressed in a mouse colon tumor. Cancer Res. 42, 1088–1093 (1982).

    CAS  PubMed  Google Scholar 

  123. Dandy, D. S., Wu, P. & Grainger, D. W. Array feature size influences nucleic acid surface capture in DNA microarrays. Proc. Natl Acad. Sci. USA 104, 8223–8228 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Keating, B. J. et al. Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies. PLoS ONE 3, e3583 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  125. DeRisi, J. et al. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat. Genet. 14, 457–460 (1996).

    Article  CAS  PubMed  Google Scholar 

  126. Alizadeh, A. A. & Staudt, L. M. Genomic-scale gene expression profiling of normal and malignant immune cells. Curr. Opin. Immunol. 12, 219–225 (2000).

    Article  CAS  PubMed  Google Scholar 

  127. Rhodes, D. R. et al. Large-scale meta-analysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression. Proc. Natl Acad. Sci. USA 101, 9309–9314 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Vora, G. J., Meador, C. E., Stenger, D. A. & Andreadis, J. D. Nucleic acid amplification strategies for DNA microarray-based pathogen detection. Appl. Environ. Microbiol. 70, 3047–3054 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Wilson, W. J. et al. Sequence-specific identification of 18 pathogenic microorganisms using microarray technology. Mol. Cell Probes 16, 119–127 (2002).

    Article  CAS  PubMed  Google Scholar 

  130. Imai, K., Kricka, L. J. & Fortina, P. Concordance study of 3 direct-to-consumer genetic-testing services. Clin. Chem. 57, 518–521 (2011).

    Article  CAS  PubMed  Google Scholar 

  131. Dolgin, E. Personalized investigation. Nat. Med. 16, 953–955 (2010).

    Article  CAS  PubMed  Google Scholar 

  132. Jia, P., Wang, L., Meltzer, H. Y. & Zhao, Z. Common variants conferring risk of schizophrenia: a pathway analysis of GWAS data. Schizophr. Res. 122, 38–42 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  133. Welter, D. et al. The NHGRI GWAS Catalog, a curated resource of SNP-trait associations. Nucleic Acids Res. 42, D1001–D1006 (2014).

    Article  CAS  PubMed  Google Scholar 

  134. Carter, N. P. Methods and strategies for analyzing copy number variation using DNA microarrays. Nat. Genet. 39, S16–S21 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Vrijenhoek, T. et al. Recurrent CNVs disrupt three candidate genes in schizophrenia patients. Am. J. Hum. Genet. 83, 504–510 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Marshall, C. R. et al. Structural variation of chromosomes in autism spectrum disorder. Am. J. Hum. Genet. 82, 477–488 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Buck, M. J. & Lieb, J. D. ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics 83, 349–360 (2004).

    Article  CAS  PubMed  Google Scholar 

  138. Liang, L. et al. A cross-platform analysis of 14,177 expression quantitative trait loci derived from lymphoblastoid cell lines. Genome Res. 23, 716–726 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Zhao, S., Fung-Leung, W. P., Bittner, A., Ngo, K. & Liu, X. Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLoS ONE 9, e78644 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  140. Holland, P. M., Abramson, R. D., Watson, R. & Gelfand, D. H. Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl Acad. Sci. USA 88, 7276–7280 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Morin, P. A. & McCarthy, M. Highly accurate SNP genotyping from historical and low-quality samples. Mol. Ecol. Notes 7, 937–946 (2007).

    Article  CAS  Google Scholar 

  142. VanGuilder, H. D., Vrana, K. E. & Freeman, W. M. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44, 619–626 (2008).

    Article  CAS  PubMed  Google Scholar 

  143. Weaver, S. et al. Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution. Methods 50, 271–276 (2010).

    Article  CAS  PubMed  Google Scholar 

  144. Sedlak, R. H., Cook, L., Cheng, A., Magaret, A. & Jerome, K. R. Clinical utility of droplet digital PCR for human cytomegalovirus. J. Clin. Microbiol. 52, 2844–2848 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  145. Kulkarni, M. M. in Current Protocols in Molecular Biology Ch. 25 (eds Ausubel, F. M. et al.) (Wiley, 2011).

    Google Scholar 

  146. Nielsen, T. et al. Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens. BMC Cancer 14, 177 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  147. Ku, B. M. et al. High-throughput profiling identifies clinically actionable mutations in salivary duct carcinoma. J. Transl. Med. 12, 299 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  148. Sailani, M. R. et al. The complex SNP and CNV genetic architecture of the increased risk of congenital heart defects in Down syndrome. Genome Res. 23, 1410–1421 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Lira, M. E. et al. Multiplexed gene expression and fusion transcript analysis to detect ALK fusions in lung cancer. J. Mol. Diagn. 15, 51–61 (2013).

    Article  CAS  PubMed  Google Scholar 

  150. Schwartz, D. C. et al. Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping. Science 262, 110–114 (1993).

    Article  CAS  PubMed  Google Scholar 

  151. Hastie, A. R. et al. Rapid genome mapping in nanochannel arrays for highly complete and accurate de novo sequence assembly of the complex Aegilops tauschii genome. PLoS ONE 8, e55864 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Cao, H. et al. Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology. Gigascience 3, 34 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  153. Pendleton, M. et al. Assembly and diploid architecture of an individual human genome via single-molecule technologies. Nat. Methods 12, 780–786 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Life Technologies. 5500 W series genetic analyzers. Life Technologies [online], https://tools.thermofisher.com/content/sfs/brochures/5500-w-series-spec-sheet.pdf (2012).

  155. Yuzuki, D. BGISEQ-500 debuts at the International Congress of Genomics 10. Next Generation Technologist [online], http://www.yuzuki.org/bgiseq-500-debut-at-the-international-congress-of-genomics-10 (24 Oct 2015).

  156. Winnick, E. Illumina launches four new systems; provides financial, Dx update at JP Morgan. GenomeWeb [online], https://www.genomeweb.com/business-news/illumina-launches-four-new-systems-provides-financial-dx-update-jp-morgan (12 Jan 2015).

  157. [No authors listed.] Illumina HiSeq 3000 Service Fees. Oregon State University [online], http://cgrb.oregonstate.edu/core/illumina-hiseq-3000/illumina-hiseq-3000-service-fees (updated 1 Jan 2016)

  158. Heger, M. Thermo Fisher launches new systems to focus on plug and play targeted sequencing. GenomeWeb [online], https://www.genomeweb.com/sequencing-technology/thermo-fisher-launches-new-systems-focus-plug-and-play-targeted-sequencing (1 Sep 2015).

  159. Ip, C. L. et al. MinION analysis and reference consortium: Phase 1 data release and analysis. F1000Research 4, 1075 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  160. Glenn, T. C. Field guide to next-generation DNA sequencers. Mol. Ecol. Resour. 11, 759–769 (2011).

    Article  CAS  PubMed  Google Scholar 

  161. Karow, J. At AGBT, 10X Genomics launches GemCode platform; shipments slated for Q2 as firm battles IP lawsuits. GenomeWeb [online], https://www.genomeweb.com/sample-prep/agbt-10x-genomics-launches-gemcode-platform-shipments-slated-q2-firm-battles-ip-lawsuits (2 Mar 2015).

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to W. Richard McCombie.

Ethics declarations

Competing interests

W.R.M. and J.D.M. have participated in Illumina sponsored meetings over the past four years and received travel reimbursement and honoraria for presenting at these events. Illumina had no role in decisions relating to the study/work to be published, data collection and analysis of data and the decision to publish. W.R.M. and J.D.M. have participated in Pacific Biosciences sponsored meetings over the past three years and received travel reimbursement for presenting at these events. S.H.G. has participated in Oxford Nanopore Technologies sponsored meetings and received travel reimbursement for presenting at these events.

Related links

PowerPoint slides

Glossary

Read

The sequence of bases from a single molecule of DNA.

Sanger sequencing

An approach in which dye-labelled normal deoxynucleotides (dNTPs) and dideoxy-modified dNTPs are mixed. A standard PCR reaction is carried out and, as elongation occurs, some strands incorporate a dideoxy-dNTP, thus terminating elongation. The strands are then separated on a gel and the terminal base label of each strand is identified by laser excitation and spectral emission analysis.

Template

A DNA fragment to be sequenced. The DNA is typically ligated to one or more adapter sequences where DNA sequencing will be initiated.

Fragmentation

The process of breaking large DNA fragments into smaller fragments. This can be achieved mechanically (by passing the DNA through a narrow passage), by sonication or enzymatically.

Clusters

Groups of DNA templates in close spatial proximity, generated either though bead-based amplification or by solid-phase amplification. Bead-based approaches rely on emulsions to maintain template isolation during amplification. Solid-phase approaches rely on the template-to-bound-adapter ratio to probabilistically bind template molecules at a sufficient distance from each other.

Flow cells

Disposable parts of a next-generation sequencing routine. Template DNA is immobilized within the flow cell where fluid reagents can be streamed into the cell and flushed away.

Rolling circle amplification

(RCA). A method of DNA amplification using a circular template. Briefly, DNA polymerase binds to a primed section of a circular DNA template. As the polymerase traverses the template, a new strand is synthesized. When the polymerase completes a full circle and encounters the double-stranded DNA (dsDNA) template, it displaces the template without degradation, thus creating a long ssDNA fragment composed of many copies of the template sequence.

One-base-encoded probes

Oligonucleotides that contain a single interrogation base in a known position. The base corresponds to a fluorescent label on each probe. The remaining bases are either degenerate (any of the four bases) or universal (unnatural bases with nonspecific hybridization), allowing the probe to interact with many different possible template sequences.

Two-base-encoded probes

Oligonucleotides that contain two adjacent interrogation bases in a known position. The bases correspond to a fluorescent label on each probe. The remaining bases are either degenerate (any of the four bases) or universal (unnatural bases with nonspecific hybridization) allowing the probe to interact with many different possible template sequences.

Colour-space

A system exclusively used by SOLiD. When a two-base-encoded probe is used, the bound label corresponds to two bases rather than one. Thus, the signal derived from a SOLiD run is in a series of colours that represent overlapping dinucleotides, rather than each colour being directly correlated to a single base. A reference-based alignment is the most efficient way to translate colour-space into base-space. For example, in the sequence ATGT the first probe will match AT, the second will match TG and the third GT. If the AT is known, then the subsequent colour order is uniquely solved as TG and GT, leading to a readout of ATGT. Final sequence deconvolution of colour-space is achieved with the knowledge of the second base identity in one round and the colour of the subsequent round in which the ligation is offset by one nucleotide, allowing for the identification of the next base.

Base-space

A system used by most next-generation sequencing platforms. When a one-base-encoded probe or a sequencing-by-synthesis approach is used, each signal is correctly correlated to a base.

Whole-genome sequencing

(WGS). Sequencing of the entire genome without using methods for sequence selection.

Two-fluorophore system

A system in which bases are discriminated by labelling Cs and Ts with a red or green fluorophore, respectively. Each A base is labelled with either a red or green fluorophore, but the two populations are mixed. During base discrimination, clusters that are either red or green are called either C or T, whereas clusters with a red and green mixed signal are called A. The G base is unlabelled, thus any cluster without a fluorophore signal is called G.

Homopolymer

A sequence run of identical bases.

Charge-coupled device

(CCD). A device composed of an integrated circuit that forms light-sensitive elements: pixels. When a photon interacts with the device, the light generates a charge that can be interpreted by an electronic device.

Integrated complementary metal-oxide-semiconductor

(CMOS). An integrated circuit design that is printed on a microchip that contains different types of semiconductor transistors to create a circuit that both uses very little power and is resistant to high levels of electronic noise.

Ion-sensitive field-effect transistor

(ISFET). A type of transistor that is sensitive to changes in ion concentration.

Single-end and paired-end sequencing

In single-end sequencing, a DNA template is sequenced only in one direction. In paired-end sequencing, a DNA template is sequenced from both sides; the forward and reverse reads may or may not overlap. A deviation in the expected genome alignment between two ends of a paired-end read can indicate astructural variation.

Structural variant

A variation larger than single-nucleotide polymorphisms (SNPs). This can include the insertion or deletion of blocks of DNA, inversions or translocations of DNA segments, and copy-number differences.

ChIP–seq

(Chromatin immunoprecipitation followed by sequencing). A method used to analyse protein interactions with DNA by combining ChIP with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.

ATAC–seq

(Assay for transposase-accessible chromatin with high-throughput sequencing). A method that uses the activity of a hyperactive transposase to cleave exposed DNA and add sequencing adapters. Regions that cannot be sequenced are inferred to be chromatin interacting.

RNA sequencing

(RNA-seq). A method of sequencing cDNA derived from RNA. This approach can be used to sequence both coding and non-coding RNA.

Real-time sequencing

A sequencing strategy used in the Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) platforms. In these approaches there is no pause after the detection of a base or series of bases, thus the sequence is derived in real-time.

Barcodes

A series of known bases added to a template molecule either through ligation or amplification. After sequencing, these barcodes can be used to identify which sample a particular read is derived from.

Zero-mode waveguides

(ZMW). Nanostructure devices used in the Pacific Biosciences (PacBio) platform. Each ZMW well (also called a waveguide) is several nanometres in diameter and is anchored to a glass substrate. The size of each well does not allow for light propagation, thus the fluorophores bound to bases can only be visualized through the glass substrate in the bottom-most portion of the well, a volume in the zeptolitre range.

Read of insert

The highest-quality single sequence for an insert, regardless of the number of passes.

Consensus sequence

In next-generation sequencing (NGS) routines that allow multiple overlapping reads from a single molecule of DNA, all related reads are aligned to each other and the most likely base at each position is determined. This process helps to overcome high, single-pass error rates. A high-quality consensus sequence derived from the circular template from Pacific Biosciences (PacBio) is called a circular consensus sequence (CCS).

Squiggle space

A system exclusively used by Oxford Nanopore Technologies (ONT). As DNA translocates through the pore, a shift in voltage occurs that is directly correlated to a k-mer within the pore. Thus, the signal derived from a nanopore run is a continuous series of voltage shifts (squiggles) that represent a series of overlapping k-mers.

K-mer

A substring within a sequence of bases of some (k) length. Currently, k-mer sizes of Oxford Nanopore Technologies (ONT) range from 3 to 6 bases.

1D and 2D reads

Oxford Nanopore Technologies (ONT) sequencing allows for both the full forward and full reverse strand of a double-stranded DNA (dsDNA) molecule to be sequenced and associated. A 1D read is the sequence of DNA bases derived from either the forward or reverse DNA strand. A 2D read is a consensus sequence derived from both the forward and the reverse reads.

BAC-by-BAC sequencing

A sequencing method where a physical map is generated from overlapping bacterial artificial chromosome (BAC) clones tiled across a chromosome. Each BAC is then fragmented and sequenced. The sequenced fragments are aligned with the knowledge of the originating BAC.

Linked reads

Reads derived from the 10X Genomics synthetic long-read platform. These are discontinuous reads each sharing the same barcode, thus they are derived from the same original long molecule.

Read cloud

The means by which the 10X Genomics platform determines a synthetic long read. Discontinuous linked reads from the same genomic region are aligned to each other. No single linked read contains the entire long sequence; however, when they are stacked, full coverage is achieved.

Polymerase reads

Contiguous sequences of nucleotides incorporated by the DNA polymerase while reading a template. These reads include sequences from adapters and can represent sequences from multiple passes around a circular template.

Single-pass

The single-molecule real-time (SMRT) sequencing approach from Pacific Biosciences (PacBio) enables a single molecule of DNA to be sequenced multiple times. A single pass is one single iteration through a molecule.

Subreads

The sequences derived from a single pass as a polymerase traverses a DNA molecule multiple times. A subread is trimmed to exclude any adapter sequence.

Whole-exome and targeted sequencing

Sequencing of only exons or other selected regions. A system of capture or amplification is used to isolate or enrich for only exons or target regions. This is done by designing probes or primers for the regions of interest.

Genome phasing

A method to identify which chromosome a DNA sequence is derived from. By examining polymorphisms, the chromosome of origin can be inferred by matching the reads that share the same variation.

Family studies

A study design in which many members of a family across several generations are sequenced. These studies are used to understand how phenotypes manifest within a particular genotype background.

Helicos Genetic Analysis System

A sequencing technology based on single nucleotide addition. Each nucleotide contains a 'virtual terminator' that prevents the incorporation of multiple nucleotides per cycle.

Fluorescence resonance energy transfer

(FRET; also known as Förster resonance energy transfer). A system in which energy can be transferred from one light-sensitive molecule to another. When the two molecules are in close proximity (≤30 nm), energy transferred between the two molecules modulates the intensity of a fluorescence signal.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Goodwin, S., McPherson, J. & McCombie, W. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet 17, 333–351 (2016). https://doi.org/10.1038/nrg.2016.49

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrg.2016.49

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