Emerging Next-Generation Sequencing Technologies

  • Matthew W. AndersonEmail author


An ideal sequencing instrument should detect all types of genomic variation including structural [single nucleotide polymorphisms (SNPs), indels, copy number variation, inversions, chromosomal rearrangements], epigenomic, and transcriptional. Long read lengths are required to enable efficient genomic assembly and accurate phasing, and the detection method must produce highly accurate base calls to minimize errors and reduce costly iterative sequencing. Finally, the system should be inexpensive, be easy to maintain and operate, and require short run times. Although “ideal” sequencing instruments do not currently exist, engineers, physicists, and biologists in both industry and academia are actively working to solve the major technical challenges facing the development of new sequencing technologies. This chapter provides a broad overview of emerging new sequencing technologies including single-molecule and nanopore approaches. Potential applications in nucleic acid analysis which will be enabled by these technological advances are also highlighted.


Sequencing DNA Nanopore Nucleotides FRET Electron microscopy Single-molecule Fluorescence PCR Pacific Biosciences Oxford Nanopore Technologies Zero-mode waveguide Polymerase Next-generation sequencing 


  1. 1.
    Walsh PS, Erlich HA, Higuchi R. Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods Appl. 1992;1:241–50.CrossRefGoogle Scholar
  2. 2.
    Cha RS, Thilly WG. Specificity, efficiency, and fidelity of PCR. PCR Methods Appl. 1993;3:S18–29.CrossRefGoogle Scholar
  3. 3.
    Shuldiner AR, Nirula A, Roth J. Hybrid DNA artifact from PCR of closely related target sequences. Nucleic Acids Res. 1989;17:4409.CrossRefGoogle Scholar
  4. 4.
    Mutter GL, Boynton KA. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies. Nucleic Acids Res. 1995;23:1411–8.CrossRefGoogle Scholar
  5. 5.
    Braslavsky I, Hebert B, Kartalov E, Quake SR. Sequence information can be obtained from single DNA molecules. Proc Natl Acad Sci U S A. 2003;100:3960–4.CrossRefGoogle Scholar
  6. 6.
    Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Braslavsky I, Causey M, Colonell J, Dimeo J, Efcavitch JW, Giladi E, Gill J, Healy J, Jarosz M, Lapen D, Moulton K, Quake SR, Steinmann K, Thayer E, Tyurina A, Ward R, Weiss H, Xie Z. Single-molecule DNA sequencing of a viral genome. Science. 2008;320:106–9.CrossRefGoogle Scholar
  7. 7.
    Pushkarev D, Neff NF, Quake SR. Single-molecule sequencing of an individual human genome. Nat Biotechnol. 2009;27:847–52.CrossRefGoogle Scholar
  8. 8.
    Ashley EA, Butte AJ, Wheeler MT, Chen R, Klein TE, Dewey FE, Dudley JT, Ormond KE, Pavlovic A, Morgan AA, Pushkarev D, Neff NF, Hudgins L, Gong L, Hodges LM, Berlin DS, Thorn CF, Sangkuhl K, Hebert JM, Woon M, Sagreiya H, Whaley R, Knowles JW, Chou MF, Thakuria JV, Rosenbaum AM, Zaranek AW, Church GM, Greely HT, Quake SR, Altman RB. Clinical assessment incorporating a personal genome. Lancet. 2010;375:1525–35.CrossRefGoogle Scholar
  9. 9.
    Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P, Thompson JF, Bowers J, Jarosz M, Milos PM. Direct RNA sequencing. Nature. 2009;461:814–8.CrossRefGoogle Scholar
  10. 10.
    Goren A, Ozsolak F, Shoresh N, Ku M, Adli M, Hart C, Gymrek M, Zuk O, Regev A, Milos PM, Bernstein BE. Chromatin profiling by directly sequencing small quantities of immunoprecipitated DNA. Nat Methods. 2010;7:47–9.CrossRefGoogle Scholar
  11. 11.
    Krol A. Direct genomics’ new clinical sequencer revives a forgotten DNA technology. Bio-IT World. Oct 29, 2015. ( Last accessed 22 March 2017.
  12. 12.
    Levene MJ, Korlach J, Turner SW, Foquet M, Craighead HG, Webb WW. Zero-mode waveguides for single-molecule analysis at high concentrations. Science. 2003;299:682–6.CrossRefGoogle Scholar
  13. 13.
    Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B, Christians F, Cicero R, Clark S, Dalal R, Dewinter A, Dixon J, Foquet M, Gaertner A, Hardenbol P, Heiner C, Hester K, Holden D, Kearns G, Kong X, Kuse R, Lacroix Y, Lin S, Lundquist P, Ma C, Marks P, Maxham M, Murphy D, Park I, Pham T, Phillips M, Roy J, Sebra R, Shen G, Sorenson J, Tomaney A, Travers K, Trulson M, Vieceli J, Wegener J, Wu D, Yang A, Zaccarin D, Zhao P, Zhong F, Korlach J, Turner S. Real-time DNA sequencing from single polymerase molecules. Science. 2009;323:133–8.CrossRefGoogle Scholar
  14. 14.
    Coupland P, Chandra T, Quail M, Reik W, Swerdlow H. Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation. BioTechniques. 2012;53:365–72.CrossRefGoogle Scholar
  15. 15.
    Korlach J, Marks PJ, Cicero RL, Gray JJ, Murphy DL, Roitman DB, Pham TT, Otto GA, Foquet M, Turner SW. Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures. Proc Natl Acad Sci U S A. 2008;105:1176–81.CrossRefGoogle Scholar
  16. 16.
    Korlach J. White paper: understanding accuracy in SMRT® sequencing. 2013. ( Last accessed 5/10/17.
  17. 17.
    Carneiro MO, Russ C, Ross MG, Gabriel SB, Nusbaum C, DePristo MA. Pacific biosciences sequencing technology for genotyping and variation discovery in human data. BMC Genomics. 2012;13:375.CrossRefGoogle Scholar
  18. 18.
    Loomis EW, Eid JS, Peluso P, Yin J, Hickey L, Rank D, McCalmon S, Hagerman RJ, Tassone F, Hagerman PJ. Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. Genome Res. 2013;23:121–8.CrossRefGoogle Scholar
  19. 19.
    Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Korlach J, Turner SW. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods. 2010;7:461–5.CrossRefGoogle Scholar
  20. 20.
    Lister R, Ecker JR. Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res. 2009;19:959–66.CrossRefGoogle Scholar
  21. 21.
    Fang G, Munera D, Friedman DI, Mandlik A, Chao MC, Banerjee O, Feng Z, Losic B, Mahajan MC, Jabado OJ, Deikus G, Clark TA, Luong K, Murray IA, Davis BM, Keren-Paz A, Chess A, Roberts RJ, Korlach J, Turner SW, Kumar V, Waldor MK, Schadt EE. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. Nat Biotechnol. 2012;30:1232–9.CrossRefGoogle Scholar
  22. 22.
    English AC, Salerno WJ, Hampton OA, Gonzaga-Jauregui C, Ambreth S, Ritter DI, Beck CR, Davis CF, Dahdouli M, Ma S, Carroll A, Veeraraghavan N, Bruestle J, Drees B, Hastie A, Lam ET, White S, Mishra P, Wang M, Han Y, Zhang F, Stankiewicz P, Wheeler DA, Reid JG, Muzny DM, Rogers J, Sabo A, Worley KC, Lupski JR, Boerwinkle E, Gibbs RA. Assessing structural variation in a personal genome – towards a human reference diploid genome. BMC Genomics. 2015;16:286–301.CrossRefGoogle Scholar
  23. 23.
    Mayor NP, Robinson J, McWhinnie AJM, Ranande S, Eng K, Midwinter W, Bultitude WP, Chin CS, Bowman B, Marks P, Braund H, Madrigal JA, Latham K, Marsh SGE. HLA typing for the next generation. PLoS One. 2015;10:e0127153.CrossRefGoogle Scholar
  24. 24.
    Cavelier L, Ameur A, Häggqvist S, Höijer I, Cahill N, Olsson-Strömberg U, Hermanson M. Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing. BMC Cancer. 2015;15:45–57.CrossRefGoogle Scholar
  25. 25.
    Vander Horn PB. Single molecule real-time sequencing on the surface of a quantum-dot nanocrystal. J Biomol Tech. 2011;22(Suppl):S9.PubMedCentralGoogle Scholar
  26. 26.
    Neely RK, Deen J, Hofkens J. Optical mapping of DNA: single-molecule-based methods for mapping genomes. Biopolymers. 2011;95:298–311.CrossRefGoogle Scholar
  27. 27.
    Schmid CW, Manning JE, Davidson N. Inverted repeat sequences in the Drosophila genome. Cell. 1975;5:159–72.CrossRefGoogle Scholar
  28. 28.
    Deininger PL, Schmid CW. An electron microscope study of the DNA sequence organization of the human genome. J Mol Biol. 1976;106:773–90.CrossRefGoogle Scholar
  29. 29.
    Bell DC, Thomas WK, Murtagh KM, Dionne CA, Graham AC, Anderson JE, Glover WR. DNA base identification by electron microscopy. Microsc Microanal. 2012;18:1049–53.CrossRefGoogle Scholar
  30. 30.
    Mankos M, Shadman K, Persson HHJ, N’Diaye AT, Schmid AK, Davis RW. A novel low energy electron microscope for DNA sequencing and surface analysis. Ultramicroscopy. 2014;145:36–49.CrossRefGoogle Scholar
  31. 31.
    Teague B, Waterman MS, Goldstein S, Potamousis K, Zhou S, Reslewic S, Sarkar D, Valouev A, Churas C, Kidd JM, Kohn S, Runnheim R, Lamers C, Forrest D, Newton MA, Eichler EE, Kent-First M, Surti U, Livny M, Schwartz DC. High-resolution human genome structure by single-molecule analysis. Proc Natl Acad Sci U S A. 2010;107:10848–53.CrossRefGoogle Scholar
  32. 32.
    Zhou S, Wei F, Nguyen J, Bechner M, Potamousis K, Goldstein S, Pape L, Mehan MR, Churas C, Pasternak S, Forrest DK, Wise R, Ware D, Wing RA, Waterman MS, Livny M, Schwartz DC. A single molecule scaffold for the maize genome. PLoS Genet. 2009;5:e1000711.CrossRefGoogle Scholar
  33. 33.
    Kasianowicz JJ, Brandin E, Branton D, Deamer DW. Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A. 1996;93:13770–3.CrossRefGoogle Scholar
  34. 34.
    Deamer D, Akeson M, Branton D. Three decades of nanopore sequencing. Nat Biotechnol. 2016;34:518–24.CrossRefGoogle Scholar
  35. 35.
    Derrington IM, Butler TZ, Collins MD, Manrao E, Pavlenok M, Niederweis M, Gundlach JH. Nanopore DNA sequencing with MspA. Proc Natl Acad Sci U S A. 2010;107:16060–5.CrossRefGoogle Scholar
  36. 36.
    Manrao EA, Derrington IM, Laszlo AH, Langford KW, Hopper MK, Gillgren N, Pavlenok M, Niederweis M, Gundlach JH. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol. 2012;30:349–53.CrossRefGoogle Scholar
  37. 37.
    Cherf GM, Lieberman KR, Rashid H, Lam CE, Karplus K, Akeson M. Automated forward and reverse ratcheting of DNA in a nanopore at 5-A precision. Nat Biotechnol. 2012;30:344–8.CrossRefGoogle Scholar
  38. 38.
    Clarke J, Wu HC, Jayasinghe L, Patel A, Reid S, Bayley H. Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol. 2009;4:265–70.CrossRefGoogle Scholar
  39. 39.
    Fuller CW, Kumar S, Porel M, Chien M, Bibillo A, Benjamin Stranges P, Dorwart M, Tao C, Li Z, Guo W, Shi S, Korenblum D, Trans A, Aguirre A, Liu E, Harada ET, Pollard J, Bhat A, Cech C, Yang A, Arnold C, Palla M, Hovis J, Chen R, Morozova I, Kalachikov S, Russo JJ, Kasianowicz JJ, Davis R, Roever S, Church GM, Ju J. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array. Proc Natl Acad Sci U S A. 2016;113:5233–8.CrossRefGoogle Scholar
  40. 40.
    Jain M, Olsen HE, Paten B, Akeson M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 2016;17:239–50.CrossRefGoogle Scholar
  41. 41.
    Laver T, Harrison J, O'Neill PA, Moore K, Farbos A, Paszkiewicz K, Studholme DJ. Assessing the performance of the Oxford Nanopore Technologies MinION. Biomol Detect Quantif. 2015;3:1–8.CrossRefGoogle Scholar
  42. 42.
    Ip CL, Loose M, Tyson JR, de Cesare M, Brown BL, Jain M, Leggett RM, Eccles DA, Zalunin V, Urban JM, Piazza P, Bowden RJ, Paten B, Mwaigwisya S, Batty EM, Simpson JT, Snutch TP, Birney E, Buck D, Goodwin S, Jansen HJ, O'Grady J, Olsen HE. MinION analysis and reference consortium: phase 1 data release and analysis. F1000Res. 2015;4:1075–110.CrossRefGoogle Scholar
  43. 43.
    Jain M, Fiddes IT, Miga KH, Olsen HE, Paten B, Akeson M. Improved data analysis for the MinION nanopore sequencer. Nat Methods. 2015;12:351–6.CrossRefGoogle Scholar
  44. 44.
    Norris AL, Workman RE, Fan Y, Eshleman JR, Timp W. Nanopore sequencing detects structural variants in cancer. Cancer Biol Ther. 2016;17:246–53.CrossRefGoogle Scholar
  45. 45.
    Bolisetty MT, Rajadinakaran G, Graveley BR. Determining exon connectivity in complex mRNAs by nanopore sequencing. Genome Biol. 2015;16:204–16.CrossRefGoogle Scholar
  46. 46.
    Quick J, Loman NJ, Duraffour S, Simpson JT, Severi E, Cowley L, Bore JA, Koundouno R, Dudas G, Mikhail A, Ouédraogo N, Afrough B, Bah A, Baum JH, Becker-Ziaja B, Boettcher JP, Cabeza-Cabrerizo M, Camino-Sánchez Á, Carter LL, Doerrbecker J, Enkirch T, García-Dorival I, Hetzelt N, Hinzmann J, Holm T, Kafetzopoulou LE, Koropogui M, Kosgey A, Kuisma E, Logue CH, Mazzarelli A, Meisel S, Mertens M, Michel J, Ngabo D, Nitzsche K, Pallasch E, Patrono LV, Portmann J, Repits JG, Rickett NY, Sachse A, Singethan K, Vitoriano I, Yemanaberhan RL, Zekeng EG, Racine T, Bello A, Sall AA, Faye O, Faye O, Magassouba N, Williams CV, Amburgey V, Winona L, Davis E, Gerlach J, Washington F, Monteil V, Jourdain M, Bererd M, Camara A, Somlare H, Camara A, Gerard M, Bado G, Baillet B, Delaune D, Nebie KY, Diarra A, Savane Y, Pallawo RB, Gutierrez GJ, Milhano N, Roger I, Williams CJ, Yattara F, Lewandowski K, Taylor J, Rachwal P, Turner DJ, Pollakis G, Hiscox JA, Matthews DA, O’Shea MK, Johnston AM, Wilson D, Hutley E, Smit E, Di Caro A, Wölfel R, Stoecker K, Fleischmann E, Gabriel M, Weller SA, Koivogui L, Diallo B, Keïta S, Rambaut A, Formenty P, Günther S, Carroll MW. Real-time portable genome sequencing for Ebola surveillance. Nature. 2016;530:228–32.CrossRefGoogle Scholar
  47. 47.
    Rosenstein JK, Wanunu M, Merchant CA, Drndic M, Shepard KL. Integrated nanopore sensing platform with sub-microsecond temporal resolution. Nat Methods. 2012;9:487–92.CrossRefGoogle Scholar
  48. 48.
    Heerema SJ, Dekker C. Graphene nanodevices for DNA sequencing. Nat Nanotech. 2016;11:127–36.CrossRefGoogle Scholar
  49. 49.
    Liu H, He J, Tang J, Liu H, Pang P, Cao D, Krstic P, Joseph S, Lindsay S, Nuckolls C. Translocation of single-stranded DNA through single-walled carbon nanotubes. Science. 2010;327:64–7.CrossRefGoogle Scholar
  50. 50.
    Balagurusamy VS, Weinger P, Ling XS. Detection of DNA hybridizations using solid-state nanopores. Nanotechnology. 2010;21:335102.CrossRefGoogle Scholar
  51. 51.
    Kowalczyk SW, Hall AR, Dekker C. Detection of local protein structures along DNA using solid-state nanopores. Nano Lett. 2010;10:324–8.CrossRefGoogle Scholar
  52. 52.
    Thompson JF, Oliver JS. Mapping and sequencing DNA using nanopores and nanodetectors. Electrophoresis. 2012;33:3429–36.CrossRefGoogle Scholar
  53. 53.
    Ohshiro T, Matsubara K, Tsutsui M, Furuhashi M, Taniguchi M, Kawai T. Single-molecule electrical random resequencing of DNA and RNA. Sci Rep. 2012;2:501.CrossRefGoogle Scholar
  54. 54.
    Ohshiro T, Umezawa Y. Complementary base-pair-facilitated electron tunneling for electrically pinpointing complementary nucleobases. Proc Natl Acad Sci U S A. 2006;103:10–4.CrossRefGoogle Scholar
  55. 55.
    Min SK, Kim WY, Cho Y, Kim KS. Fast DNA sequencing with a graphene-based nanochannel device. Nat Nanotechnol. 2011;6:162–5.CrossRefGoogle Scholar
  56. 56.
    Morin TJ, Shropshire T, Liu X, Briggs K, Huynh C, Tabard-Cossa V, Wang H, Dunbar WB. Nanopore-based target sequence detection. PLoS One. 2016;11:e0154426.CrossRefGoogle Scholar
  57. 57.
    Anderson MW, Schrijver I. Next generation DNA sequencing and the future of genomic medicine. Genes. 2010;1:38–69.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Diagnostic Laboratories, BloodCenter of Wisconsin, part of VersitiMilwaukeeUSA

Personalised recommendations