Skip to main content

Thank you for visiting 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.

Long-read sequencing for rare human genetic diseases


During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. 1.

    Royer-Pokora B, Kunkel LM, Monaco AP, Goff SC, Newburger PE, Baehner RL, et al. Cloning the gene for an inherited human disorder–chronic granulomatous disease–on the basis of its chromosomal location. Nature. 1986;322:32–8.

  2. 2.

    Monaco AP, Neve RL, Colletti-Feener C, Bertelson CJ, Kurnit DM, Kunkel LM. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature. 1986;323:646–50.

  3. 3.

    Clark MM, Stark Z, Farnaes L, Tan TY, White SM, Dimmock D, et al. Meta-analysis of the diagnostic and clinical utility of genome and exome sequencing and chromosomal microarray in children with suspected genetic diseases. NPJ Genom Med. 2018;3:16.

  4. 4.

    Harada N, Hatchwell E, Okamoto N, Tsukahara M, Kurosawa K, Kawame H, et al. Subtelomere specific microarray based comparative genomic hybridisation: a rapid detection system for cryptic rearrangements in idiopathic mental retardation. J Med Genet. 2004;41:130–6.

  5. 5.

    Neerman N, Faust G, Meeks N, Modai S, Kalfon L, Falik-Zaccai T, et al. A clinically validated whole genome pipeline for structural variant detection and analysis. BMC Genomics. 2019;20:545.

  6. 6.

    Sone J, Fujita A, Mizuguchi T, Hamanaka K, Mori K, Koike H, et al. Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease. Nat Genet. 2019;51:1215–1221.

  7. 7.

    Merker JD, Wenger AM, Sneddon T, Grove M, Zappala Z, Fresard L, et al. Long-read genome sequencing identifies causal structural variation in a Mendelian disease. Genet Med. 2018;20:159–63.

  8. 8.

    Mizuguchi T, Suzuki T, Abe C, Umemura A, Tokunaga K, Kawai Y, et al. A 12-kb structural variation in progressive myoclonic epilepsy was newly identified by long-read whole-genome sequencing. J Hum Genet. 2019;64:359–68.

  9. 9.

    Mizuguchi T, Toyota T, Adachi H, Miyake N, Matsumoto N, Miyatake S. Mizuguchi T, Toyota T, Adachi H, Miyake N, Matsumoto N, Miyatake S. Detecting a long insertion variant in SAMD12 by SMRT sequencing: implications of long-read whole-genome sequencing for repeat expansion diseases. J Hum Genet. 2018;64:191–97.

  10. 10.

    Zeng S, Zhang MY, Wang XJ, Hu ZM, Li JC, Li N, et al. Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy. J Med Genet. 2019;56:265–70.

  11. 11.

    Sanchis-Juan A, Stephens J, French CE, Gleadall N, Megy K, Penkett C, et al. Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing. Genome Med. 2018;10:95.

  12. 12.

    Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999;27:573–80.

  13. 13.

    Mitsuhashi S, Frith MC, Mizuguchi T, Miyatake S, Toyota T, Adachi H, et al. Tandem-genotypes: robust detection of tandem repeat expansions from long DNA reads. Genome Biol. 2019;20:58.

  14. 14.

    Weber JL, Wong C. Mutation of human short tandem repeats. Hum Mol Genet. 1993;2:1123–8.

  15. 15.

    Brinkmann B, Klintschar M, Neuhuber F, Huhne J, Rolf B. Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat. Am J Hum Genet. 1998;62:1408–15.

  16. 16.

    Tang H, Kirkness EF, Lippert C, Biggs WH, Fabani M, Guzman E, et al. Profiling of Short-Tandem-Repeat Disease Alleles in 12,632 Human Whole Genomes. Am J Hum Genet. 2017;101:700–15.

  17. 17.

    Deenen JC, Arnts H, van der Maarel SM, Padberg GW, Verschuuren JJ, Bakker E, et al. Population-based incidence and prevalence of facioscapulohumeral dystrophy. Neurology. 2014;83:1056–9.

  18. 18.

    Brook JD, McCurrach ME, Harley HG, Buckler AJ, Church D, Aburatani H, et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell. 1992;68:799–808.

  19. 19.

    Lemmers RJ, van der Vliet PJ, Klooster R, Sacconi S, Camano P, Dauwerse JG, et al. A unifying genetic model for facioscapulohumeral muscular dystrophy. Science. 2010;329:1650–3.

  20. 20.

    Cumming SA, Hamilton MJ, Robb Y, Gregory H, McWilliam C, Cooper A, et al. De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1. Eur J Hum Genet. 2018;26:1635–47.

  21. 21.

    Mitsuhashi S, Nakagawa S, Takahashi Ueda M, Imanishi T, Frith MC, Mitsuhashi H. Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy. Sci Rep. 2017;7:14789.

  22. 22.

    Kremer EJ, Pritchard M, Lynch M, Yu S, Holman K, Baker E, et al. Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science. 1991;252:1711–4.

  23. 23.

    Loomis EW, Eid JS, Peluso P, Yin J, Hickey L, Rank D, et al. Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. Genome Res. 2013;23:121–8.

  24. 24.

    Ardui S, Race V, de Ravel T, Van Esch H, Devriendt K, Matthijs G, et al. Detecting AGG Interruptions in Females With a FMR1 Premutation by Long-Read Single-Molecule Sequencing: A 1 Year Clinical Experience. Front Genet. 2018;9:150.

  25. 25.

    Ishiura H, Doi K, Mitsui J, Yoshimura J, Matsukawa MK, Fujiyama A, et al. Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy. Nat Genet. 2018;50:581–90.

  26. 26.

    Hamada M, Ono Y, Asai K, Frith MC. Training alignment parameters for arbitrary sequencers with LAST-TRAIN. Bioinformatics. 2017;33:926–28.

  27. 27.

    Suzuki IK, Gacquer D, Van Heurck R, Kumar D, Wojno M, Bilheu A, et al. Human-Specific NOTCH2NL Genes Expand Cortical Neurogenesis through Delta/Notch Regulation. Cell. 2018;173:1370–84.e16.

  28. 28.

    Alkan C, Coe BP, Eichler EE. Genome structural variation discovery and genotyping. Nat Rev Genet. 2011;12:363–76.

  29. 29.

    Kloosterman WP, Hochstenbach R. Deciphering the pathogenic consequences of chromosomal aberrations in human genetic disease. Mol Cytogenet. 2014;7:100.

  30. 30.

    Pounraja VK, Jayakar G, Jensen M, Kelkar N, Girirajan S. A machine-learning approach for accurate detection of copy number variants from exome sequencing. Genome Res. 2019;29:1134–43.

  31. 31.

    Collins RL, Brand H, Redin CE, Hanscom C, Antolik C, Stone MR, et al. Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome. Genome Biol. 2017;18:36.

  32. 32.

    Stancu MC, van Roosmalen MJ, Renkens I, Nieboer MM, Middelkamp S, de Ligt J, et al. Mapping and phasing of structural variation in patient genomes using nanopore sequencing. Nat Commun. 2017;8:1326.

  33. 33.

    Dutta UR, Rao SN, Pidugu VK, Vineeth VS, Bhattacherjee A, Bhowmik AD, et al. Breakpoint mapping of a novel de novo translocation t(X;20)(q11.1;p13) by positional cloning and long read sequencing. Genomics. 2018;111:1108–1114.

  34. 34.

    Reiner J, Pisani L, Qiao W, Singh R, Yang Y, Shi L, et al. Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet-Biedl Syndrome 9 (BBS9) deletion. NPJ Genom Med. 2018;3.

  35. 35.

    Miao H, Zhou J, Yang Q, Liang F, Wang D, Ma N, et al. Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis. Hereditas. 2018;155:32.

  36. 36.

    Sato N, Amino T, Kobayashi K, Asakawa S, Ishiguro T, Tsunemi T, et al. Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats containing (TGGAA)n. Am J Hum Genet. 2009;85:544–57.

  37. 37.

    McFarland KN, Liu J, Landrian I, Godiska R, Shanker S, Yu F, et al. SMRT Sequencing of Long Tandem Nucleotide Repeats in SCA10 Reveals Unique Insight of Repeat Expansion Structure. PLoS ONE. 2015;10:e0135906.

  38. 38.

    Wenzel A, Altmueller J, Ekici AB, Popp B, Stueber K, Thiele H, et al. Single molecule real time sequencing in ADTKD-MUC1 allows complete assembly of the VNTR and exact positioning of causative mutations. Sci Rep. 2018;8:4170.

  39. 39.

    Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–78.

  40. 40.

    Wenger AM, Peluso P, Rowell WJ, Chang PC, Hall RJ, Concepcion GT, et al. Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. NatBiotechnol. 2019.

  41. 41.

    Hoijer I, Tsai YC, Clark TA, Kotturi P, Dahl N, Stattin EL, et al. Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing. Hum Mutat. 2018;39, 1262–72 (2018).

  42. 42.

    Ebbert MTW, Farrugia SL, Sens JP, Jansen-West K, Gendron TF, Prudencio M, et al. Long-read sequencing across the C9orf72 'GGGGCC' repeat expansion: implications for clinical use and genetic discovery efforts in human disease. Mol Neurodegener. 2018;13:46.

  43. 43.

    Hafford-Tear NJ, Tsai YC, Sadan AN, Sanchez-Pintado B, Zarouchlioti C, Maher GJ, et al. CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat. Genet Med. 2019;21:2092–2102.

  44. 44.

    Schule B, McFarland KN, Lee K, Tsai YC, Nguyen KD, Sun C, et al. Parkinson's disease associated with pure ATXN10 repeat expansion. NPJParkinsons Dis. 2017;3:27.

  45. 45.

    Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–66.

  46. 46.

    Hancks DC, Kazazian HH Jr., Active human retrotransposons: variation and disease. Curr Opin Genet Dev. 2012;22:191–203.

  47. 47.

    Goncalves A, Oliveira J, Coelho T, Taipa R, Melo-Pires M, Sousa M, et al. Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene. Genes (Basel). 2017;8.

  48. 48.

    Aneichyk T, Hendriks WT, Yadav R, Shin D, Gao D, Vaine CA, et al. Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly. Cell. 2018;172:897–909.e21.

  49. 49.

    Baillie JK, Barnett MW, Upton KR, Gerhardt DJ, Richmond TA, De Sapio F, et al. Somatic retrotransposition alters the genetic landscape of the human brain. Nature. 2011;479:534–7.

Download references


We thank Rebecca Porter, Ph.D. and Angela Morben, DVM, ELS from Edanz Group ( for editing a draft of this manuscript. This work was supported by JSPS KAKENHI under grant numbers 19K07977 and JP17H01539 and by AMED under grant numbers JP18ek0109280, JP18dm0107090, JP18ek0109301, JP18ek0109348, and JP18kk020501.

Author information



Corresponding author

Correspondence to Satomi Mitsuhashi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mitsuhashi, S., Matsumoto, N. Long-read sequencing for rare human genetic diseases. J Hum Genet 65, 11–19 (2020).

Download citation

Further reading


Quick links