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


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.


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



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.


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)


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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

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