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

  • Nature Reviews Genetics volume 17, pages 333351 (2016)
  • doi:10.1038/nrg.2016.49
  • Download Citation


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.

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  1. 1.

    & The structure of DNA. Cold Spring Harb. Symp. Quant. Biol. 18, 123–131 (1953).

  2. 2.

    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.

  3. 3.

    DNA sequencing costs: data from the NHGRI Genome Sequencing Program (GSP). National Human Genome Research Institute [online], (updated 15 Jan 2016).

  4. 4.

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

  5. 5.

    Veritas Genetics. Veritas genetics launches $999 whole genome and sets new standard for genetic testing — Press Release. Veritas Genetics [online], (updated 4 Mar 2016).

  6. 6.

    Veritas Genetics. Veritas genetics breaks $1,000 whole genome barrier — Press Release. Veritas Genetics [online], (29 Sep 2015).

  7. 7.

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

  8. 8.

    , , , & Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl Acad. Sci. USA 100, 8817–8822 (2003).

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

    , , , & BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies. Nucleic Acids Res. 34, e22 (2006).

  13. 13.

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

  14. 14.

    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).

  15. 15.

    , , & DNA ligases: structure, reaction mechanism, and function. Chem. Rev. 106, 687–699 (2006).

  16. 16.

    , , & A ligase-mediated gene detection technique. Science 241, 1077–1080 (1988).

  17. 17.

    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.

  18. 18.

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

  19. 19.

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

  20. 20.

    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).

  21. 21.

    DNA sequencing market will exceed $20 billion, says Illumina CEO Jay Flatley. Forbes [online], (29 Apr 2015).

  22. 22.

    Qiagen launches GeneReader NGS System at AMP; presents performance evaluation by broad. GenomeWeb [online], (4 Nov 2015).

  23. 23.

    & Methods of producing and sequencing modified polynucleotides. US Patent 8058030 (2011).

  24. 24.

    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.

  25. 25.

    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.

  26. 26.

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

  27. 27.

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

  28. 28.

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

  29. 29.

    , , & Comparing platforms for C. elegans mutant identification using high-throughput whole-genome sequencing. PLoS ONE 3, e4012 (2008).

  30. 30.

    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).

  31. 31.

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

  32. 32.

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

  33. 33.

    BGI. Revolocity Whole Genome Sequencing Service — Press Release. BGI [online], (2015).

  34. 34.

    BGI halts revolocity launch, cuts complete genomics staff as part of strategic shift. GenomeWeb [online], (23 Nov 2015).

  35. 35.

    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.

  36. 36.

    , , & Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res. 36, e105 (2008).

  37. 37.

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

  38. 38.

    , & Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and genome analyzer systems. Genome Biol. 12, R112 (2011).

  39. 39.

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

  40. 40.

    , , , & Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nat. Methods 5, 865–867 (2008).

  41. 41.

    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.

  42. 42.

    , , , & 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).

  43. 43.

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

  44. 44.

    , & 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.

  45. 45.

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

  46. 46.

    Qiagen. Oncology insights enabled by knowledge base-guided panel design and the seamless workflow of the GeneReader NGS system — Press Release. Qiagen [online], (2016).

  47. 47.

    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).

  48. 48.

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

  49. 49.

    GenomeWeb. Roche shutting down 454 sequencing business. GenomeWeb [online], (15 Oct 2015).

  50. 50.

    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).

  51. 51.

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

  52. 52.

    Life Technologies. Ion semiconductor sequencing uniquely enables both accurate long reads and paired-end sequencing. Life Technologies [online], (2011)

  53. 53.

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

  54. 54.

    & Copy-number variation and association studies of human disease. Nat. Genet. 39, S37–S42 (2007).

  55. 55.

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

  56. 56.

    & Structural variation in the human genome and its role in disease. Annu. Rev. Med. 61, 437–455 (2010).

  57. 57.

    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.

  58. 58.

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

  59. 59.

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

  60. 60.

    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.

  61. 61.

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

  62. 62.

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

  63. 63.

    , & Assembly of large genomes using second-generation sequencing. Genome Res. 20, 1165–1173 (2010).

  64. 64.

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

  65. 65.

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

  66. 66.

    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).

  67. 67.

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

  68. 68.

    , & The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms. BMC Genomics 15, 720 (2014).

  69. 69.

    PacBio launches higher-throughput, lower-cost single-molecule sequencing system. GenomeWeb [online], (01 Oct 2015).

  70. 70.

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

  71. 71.

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

  72. 72.

    10X Genomics, Pacific Biosciences provide business updates at JP Morgan Healthcare Conference. GenomeWeb [online], (13 Jan 2016).

  73. 73.

    & 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.

  74. 74.

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

  75. 75.

    & Mammary development meets cancer genomics. Nat. Med. 15, 842–844 (2009).

  76. 76.

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

  77. 77.

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

  78. 78.

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

  79. 79.

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

  80. 80.

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

  81. 81.

    U.S. to develop DNA study of one million people. MIT Technology Review [online], (30 Jan 2015).

  82. 82.

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

  83. 83.

    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.

  84. 84.

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

  85. 85.

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

  86. 86.

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

  87. 87.

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

  88. 88.

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

  89. 89.

    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).

  90. 90.

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

  91. 91.

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

  92. 92.

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

  93. 93.

    , & Nanopores discriminate among five C5-cytosine variants in DNA. J. Am. Chem. Soc. 136, 16582–16587 (2014).

  94. 94.

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

  95. 95.

    , & An assessment of the sequence gaps: unfinished business in a finished human genome. Nat. Rev. Genet. 5, 345–354 (2004).

  96. 96.

    , & Genetic variation and the de novo assembly of human genomes. Nat. Rev. Genet. 16, 627–640 (2015).

  97. 97.

    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.

  98. 98.

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

  99. 99.

    , , & Haplotype-resolved genome sequencing: experimental methods and applications. Nat. Rev. Genet. 16, 344–358 (2015).

  100. 100.

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

  101. 101.

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

  102. 102.

    , , & A single-molecule long-read survey of the human transcriptome. Nat. Biotechnol. 31, 1009–1014 (2013).

  103. 103.

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

  104. 104.

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

  105. 105.

    GenomeWeb. White House announces efforts to accelerate precision medicine initiative. GenomeWeb [online], (25 Feb 2016).

  106. 106.

    Illumina. Illumina forms new company to enable early cancer detection via blood-based screening — Press Release. Illumina [online], (10 Jan 2016).

  107. 107.

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

  108. 108.

    & Bioinformatics challenges of new sequencing technology. Trends Genet. 24, 142–149 (2008).

  109. 109.

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

  110. 110.

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

  111. 111.

    & Whole genome sequencing as a diagnostic test: challenges and opportunities. Clin. Chem. 60, 724–733 (2014).

  112. 112.

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

  113. 113.

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

  114. 114.

    China's Direct Genomics unveils new targeted NGS system based on Helicos Tech for clinical use. GenomeWeb [online], (27 Oct 2015).

  115. 115.

    Oxford Nanopore presents details on new high-throughput sequencer, improvements to MinIon. GenomeWeb [online], (16 Sep 2014).

  116. 116.

    Sigma-Aldrich enters co-marketing agreement with GenapSys for Genius sequencer. GenomeWeb [online], (1 Jul 2015).

  117. 117.

    Roche. Roche acquires Genia Technologies to strengthen next generation sequencing pipeline — Press Release. Roche [online], (2 Jun 2014).

  118. 118.

    Illumina unveils mini targeted sequencer, semiconductor sequencing project at JP Morgan Conference. GenomeWeb [online], (1 Jan 2016).

  119. 119.

    NanoString. NanoString Technologies presents proof-of-concept data for novel massively parallel single molecule sequencing chemistry at AGBT meeting — Press Release. NanoString [online], (11 Feb 2016).

  120. 120.

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

  121. 121.

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

  122. 122.

    & Cloning and screening of sequences expressed in a mouse colon tumor. Cancer Res. 42, 1088–1093 (1982).

  123. 123.

    , & Array feature size influences nucleic acid surface capture in DNA microarrays. Proc. Natl Acad. Sci. USA 104, 8223–8228 (2007).

  124. 124.

    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).

  125. 125.

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

  126. 126.

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

  127. 127.

    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).

  128. 128.

    , , & Nucleic acid amplification strategies for DNA microarray-based pathogen detection. Appl. Environ. Microbiol. 70, 3047–3054 (2004).

  129. 129.

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

  130. 130.

    , & Concordance study of 3 direct-to-consumer genetic-testing services. Clin. Chem. 57, 518–521 (2011).

  131. 131.

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

  132. 132.

    , , & Common variants conferring risk of schizophrenia: a pathway analysis of GWAS data. Schizophr. Res. 122, 38–42 (2010).

  133. 133.

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

  134. 134.

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

  135. 135.

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

  136. 136.

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

  137. 137.

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

  138. 138.

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

  139. 139.

    , , , & Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLoS ONE 9, e78644 (2014).

  140. 140.

    , , & 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).

  141. 141.

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

  142. 142.

    , & Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44, 619–626 (2008).

  143. 143.

    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).

  144. 144.

    , , , & Clinical utility of droplet digital PCR for human cytomegalovirus. J. Clin. Microbiol. 52, 2844–2848 (2014).

  145. 145.

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

  146. 146.

    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).

  147. 147.

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

  148. 148.

    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).

  149. 149.

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

  150. 150.

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

  151. 151.

    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).

  152. 152.

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

  153. 153.

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

  154. 154.

    Life Technologies. 5500 W series genetic analyzers. Life Technologies [online], (2012).

  155. 155.

    BGISEQ-500 debuts at the International Congress of Genomics 10. Next Generation Technologist [online], (24 Oct 2015).

  156. 156.

    Illumina launches four new systems; provides financial, Dx update at JP Morgan. GenomeWeb [online], (12 Jan 2015).

  157. 157.

    [No authors listed.] Illumina HiSeq 3000 Service Fees. Oregon State University [online], (updated 1 Jan 2016)

  158. 158.

    Thermo Fisher launches new systems to focus on plug and play targeted sequencing. GenomeWeb [online], (1 Sep 2015).

  159. 159.

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

  160. 160.

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

  161. 161.

    At AGBT, 10X Genomics launches GemCode platform; shipments slated for Q2 as firm battles IP lawsuits. GenomeWeb [online], (2 Mar 2015).

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Author information


  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

    • Sara Goodwin
    •  & W. Richard McCombie
  2. Department of Biochemistry and Molecular Medicine; and the Comprehensive Cancer Center, University of California, Davis, California 95817, USA.

    • John D. McPherson


  1. Search for Sara Goodwin in:

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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.

Corresponding author

Correspondence to W. Richard McCombie.



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.


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


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.


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.


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.


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.


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.


(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.


(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.


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.


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.


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.


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.