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

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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.

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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.

Author information

Author notes

  1. Jun Yin

    Present address: Dendrite Morphogenesis and Plasticity Unit, National Institute of Neurological Disorders and Stroke, Bethesda, MD, 20892, USA

  2. John S. Eid

    Present address: Whole Biome, Inc., San Francisco, CA, 94107, USA

  3. Erick W. Loomis

    Present address: Faculty of Medicine, Department of Surgery & Cancer, Institute of Reproductive and Developmental Biology, Hammersmith Campus Imperial College London, London, UK

Authors and Affiliations

  1. Pacific Biosciences, Menlo Park, CA, 94025, USA

    Thang T. Pham, John S. Eid, Regina Lam, Stephen W. Turner & Jeremiah W. Hanes

  2. Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA, 95616, USA

    Jun Yin, Evan Adams, Erick W. Loomis, Jun Yi Wang & Paul J. Hagerman

Authors
  1. Thang T. Pham
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  2. Jun Yin
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  3. John S. Eid
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  4. Evan Adams
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  5. Regina Lam
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  7. Erick W. Loomis
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  8. Jun Yi Wang
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  9. Paul J. Hagerman
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  10. Jeremiah W. Hanes
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Corresponding author

Correspondence to Jeremiah W. Hanes.

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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.).

Additional information

Communicated by S. Hohmann.

T. T. Pham and J. Yin contributed equally to this manuscript.

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Pham, T.T., Yin, J., Eid, J.S. et al. Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications. Mol Genet Genomics 291, 1491–1504 (2016). https://doi.org/10.1007/s00438-016-1167-2

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  • DOI: https://doi.org/10.1007/s00438-016-1167-2

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