Advertisement

Molecular Genetics and Genomics

, Volume 291, Issue 3, pp 1491–1504 | Cite as

Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications

  • Thang T. Pham
  • Jun Yin
  • John S. Eid
  • Evan Adams
  • Regina Lam
  • Stephen W. Turner
  • Erick W. Loomis
  • Jun Yi Wang
  • Paul J. Hagerman
  • Jeremiah W. HanesEmail author
Methods Paper

Abstract

A gene-level targeted enrichment method for direct detection of epigenetic modifications is described. The approach is demonstrated on the CGG-repeat region of the FMR1 gene, for which large repeat expansions, hitherto refractory to sequencing, are known to cause fragile X syndrome. In addition to achieving a single-locus enrichment of nearly 700,000-fold, the elimination of all amplification steps removes PCR-induced bias in the repeat count and preserves the native epigenetic modifications of the DNA. In conjunction with the single-molecule real-time sequencing approach, this enrichment method enables direct readout of the methylation status and the CGG repeat number of the FMR1 allele(s) for a clonally derived cell line. The current method avoids potential biases introduced through chemical modification and/or amplification methods for indirect detection of CpG methylation events.

Keywords

Targeted enrichment Single molecule sequencing FMR1 Fragile X syndrome Epigenetic modification Tandem repeats 

Notes

Acknowledgments

The authors wish to thank the entire staff at Pacific Biosciences, in particular Leewin Chern for PCR experiments, Karl Voss for helpful discussions, and the families that have contributed to our fragile X research.

Compliance with ethical standards

Conflict of interest

Thang T. Pham, John S. Eid, Regina Lam, Stephen W. Turner and Jeremiah W. Hanes were employed at Pacific Biosciences (manufacturer of the PacBio RS II DNA sequencing instrument used in this study) throughout the course of this study. Paul J. Hagerman is a nonremunerative collaborator with Pacific Biosciences and with Roche Diagnostics; he also holds a patent for PCR-based methods for sizing CGG repeats. All other authors declare no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This work was supported by the National Institutes of Health (R01HD040661 to P.J.H.).

Supplementary material

438_2016_1167_MOESM1_ESM.docx (964 kb)
Supplementary material 1 (DOCX 964 kb)

References

  1. Arocena DG, Iwahashi CK, Won N, Beilina A, Ludwig AL, Tassone F, Schwartz PH, Hagerman PJ (2005) Induction of inclusion formation and disruption of lamin A/C structure by premutation CGG-repeat RNA in human cultured neural cells. Hum Mol Genet 14(23):3661–3671CrossRefPubMedGoogle Scholar
  2. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J (2013) Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10(6):563–569CrossRefPubMedGoogle Scholar
  3. Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloglu A, Ozen S, Sanjad S, Nelson-Williams C, Farhi A, Mane S, Lifton RP (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA 106(45):19096–19101CrossRefPubMedPubMedCentralGoogle Scholar
  4. Dahl F, Gullberg M, Stenberg J, Landegren U, Nilsson M (2005) Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments. Nucleic Acids Res 33(8):e71CrossRefPubMedPubMedCentralGoogle Scholar
  5. Deaton AM, Bird A (2011) CpG islands and the regulation of transcription. Genes Dev 25(10):1010–1022CrossRefPubMedPubMedCentralGoogle Scholar
  6. Evans-Galea MV, Hannan AJ, Carrodus N, Delatycki MB, Saffery R (2013) Epigenetic modifications in trinucleotide repeat diseases. Trends Mol Med 19(11):655–663CrossRefPubMedGoogle Scholar
  7. Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Korlach J, Turner SW (2010) Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7(6):461–465CrossRefPubMedPubMedCentralGoogle Scholar
  8. Fu Y, He C (2012) Nucleic acid modifications with epigenetic significance. Curr Opin Chem Biol 16(5–6):516–524CrossRefPubMedPubMedCentralGoogle Scholar
  9. Gallagher A, Hallahan B (2012) Fragile X-associated disorders: a clinical overview. J Neurol 259(3):401–413CrossRefPubMedGoogle Scholar
  10. Hagerman P (2013) Fragile X-associated tremor/ataxia syndrome (FXTAS): pathology and mechanisms. Acta Neuropathol 126(1):1–19CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hagerman R, Hagerman P (2013) Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome. Lancet Neurol 12(8):786–798CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hagerman R, Hoem G, Hagerman P (2010) Fragile X and autism: intertwined at the molecular level leading to targeted treatments. Mol Autism 1(1):12CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hutchison CA 3rd, Smith HO, Pfannkoch C, Venter JC (2005) Cell-free cloning using phi29 DNA polymerase. Proc Natl Acad Sci U S A 102(48):17332–17336CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kieleczawa J (2006) Fundamentals of sequencing of difficult templates—an overview. J Biomol Tech 17(3):207–217PubMedPubMedCentralGoogle Scholar
  15. Lister R, Mukamel EA, Nery JR, Urich M, Puddifoot CA, Johnson ND, Lucero J, Huang Y, Dwork AJ, Schultz MD, Yu M, Tonti-Filippini J, Heyn H, Hu S, Wu JC, Rao A, Esteller M, He C, Haghighi FG, Sejnowski TJ, Behrens MM, Ecker JR (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341(6146):1237905CrossRefPubMedPubMedCentralGoogle Scholar
  16. Loomis EW, Eid JS, Peluso P, Yin J, Hickey L, Rank D, McCalmon S, Hagerman RJ, Tassone F, Hagerman PJ (2013) Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. Genome Res 23(1):121–128CrossRefPubMedPubMedCentralGoogle Scholar
  17. Maga G, Villani G, Crespan E, Wimmer U, Ferrari E, Bertocci B, Hubscher U (2007) 8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins. Nature 447(7144):606–608CrossRefPubMedGoogle Scholar
  18. Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J, Turner DJ (2010) Target-enrichment strategies for next-generation sequencing. Nat Methods 7(2):111–118CrossRefPubMedGoogle Scholar
  19. Marmolino D (2011) Friedreich’s ataxia: past, present and future. Brain Res Rev 67(1–2):311–330CrossRefPubMedGoogle Scholar
  20. Mirkin SM (2007) Expandable DNA repeats and human disease. Nature 447(7147):932–940CrossRefPubMedGoogle Scholar
  21. Murray IA, Clark TA, Morgan RD, Boitano M, Anton BP, Luong K, Fomenkov A, Turner SW, Korlach J, Roberts RJ (2012) The methylomes of six bacteria. Nucleic Acids Res 40(22):11450–11462CrossRefPubMedPubMedCentralGoogle Scholar
  22. Mutter GL, Boynton KA (1995) PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies. Nucleic Acids Res 23(8):1411–1418CrossRefPubMedPubMedCentralGoogle Scholar
  23. Nelson DL, Orr HT, Warren ST (2013) The unstable repeats–three evolving faces of neurological disease. Neuron 77(5):825–843CrossRefPubMedPubMedCentralGoogle Scholar
  24. Primerano B, Tassone F, Hagerman RJ, Hagerman P, Amaldi F, Bagni C (2002) Reduced FMR1 mRNA translation efficiency in fragile X patients with premutations. RNA 8(12):1482–1488PubMedPubMedCentralGoogle Scholar
  25. Shen L, Zhang Y (2013) 5-Hydroxymethylcytosine: generation, fate, and genomic distribution. Curr Opin Cell Biol 25(3):289–296CrossRefPubMedPubMedCentralGoogle Scholar
  26. Shen L, Wu H, Diep D, Yamaguchi S, D’Alessio AC, Fung HL, Zhang K, Zhang Y (2013) Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153(3):692–706CrossRefPubMedPubMedCentralGoogle Scholar
  27. So A, Pel J, Rajan S, Marziali A (2010) Efficient genomic DNA extraction from low target concentration bacterial cultures using SCODA DNA extraction technology. Cold Spring Harb Protoc 2010(10): pdb prot5506Google Scholar
  28. Song CX, He C (2013) Potential functional roles of DNA demethylation intermediates. Trends Biochem Sci 38(10):480–484CrossRefPubMedPubMedCentralGoogle Scholar
  29. Song CX, Szulwach KE, Dai Q, Fu Y, Mao SQ, Lin L, Street C, Li Y, Poidevin M, Wu H, Gao J, Liu P, Li L, Xu GL, Jin P, He C (2013) Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153(3):678–691CrossRefPubMedPubMedCentralGoogle Scholar
  30. Taddei F, Hayakawa H, Bouton M, Cirinesi A, Matic I, Sekiguchi M, Radman M (1997) Counteraction by MutT protein of transcriptional errors caused by oxidative damage. Science 278(5335):128–130CrossRefPubMedGoogle Scholar
  31. Tassone F, Hagerman RJ, Taylor AK, Gane LW, Godfrey TE, Hagerman PJ (2000) Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome. Am J Hum Genet 66(1):6–15CrossRefPubMedPubMedCentralGoogle Scholar
  32. Teer JK, Bonnycastle LL, Chines PS, Hansen NF, Aoyama N, Swift AJ, Abaan HO, Albert TJ, Program NCS, Margulies EH, Green ED, Collins FS, Mullikin JC, Biesecker LG (2010) Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing. Genome Res 20(10):1420–1431CrossRefPubMedPubMedCentralGoogle Scholar
  33. Travers KJ, Chin CS, Rank DR, Eid JS, Turner SW (2010) A flexible and efficient template format for circular consensus sequencing and SNP detection. Nucleic Acids Res 38(15):e159CrossRefPubMedPubMedCentralGoogle Scholar
  34. Udd B, Krahe R (2012) The myotonic dystrophies: molecular, clinical, and therapeutic challenges. Lancet Neurol 11(10):891–905CrossRefPubMedGoogle Scholar
  35. Verkerk AJ, Pieretti M, Sutcliffe JS, Fu YH, Kuhl DP, Pizzuti A, Reiner O, Richards S, Victoria MF, Zhang FP et al (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65(5):905–914CrossRefPubMedGoogle Scholar
  36. Walker FO (2007) Huntington’s disease. Lancet 369(9557):218–228CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Thang T. Pham
    • 1
  • Jun Yin
    • 2
    • 3
  • John S. Eid
    • 1
    • 4
  • Evan Adams
    • 2
  • Regina Lam
    • 1
  • Stephen W. Turner
    • 1
  • Erick W. Loomis
    • 2
    • 5
  • Jun Yi Wang
    • 2
  • Paul J. Hagerman
    • 2
  • Jeremiah W. Hanes
    • 1
    Email author
  1. 1.Pacific BiosciencesMenlo ParkUSA
  2. 2.Department of Biochemistry and Molecular MedicineUniversity of California, Davis, School of MedicineDavisUSA
  3. 3.Dendrite Morphogenesis and Plasticity UnitNational Institute of Neurological Disorders and StrokeBethesdaUSA
  4. 4.Whole Biome, Inc.San FranciscoUSA
  5. 5.Faculty of Medicine, Department of Surgery & CancerInstitute of Reproductive and Developmental Biology, Hammersmith Campus Imperial College LondonLondonUK

Personalised recommendations