Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications
- 1.2k Downloads
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.
KeywordsTargeted 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.
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.).
- 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
- 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
- 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
- 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
- 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