title: Single-locus enrichment without amplification for sequencing and direct detection of epigenetic modifications.
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.
authors.affiliation: Pacific Biosciences, Menlo Park, CA, 94025, USA.
authors.forename: Thang T
authors.initials: TT
authors.lastname: Pham
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA, 95616, USA.|Dendrite Morphogenesis and Plasticity Unit, National Institute of Neurological Disorders and Stroke, Bethesda, MD, 20892, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.affiliation: Pacific Biosciences, Menlo Park, CA, 94025, USA.|Whole Biome, Inc., San Francisco, CA, 94107, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Eid
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA, 95616, USA.
authors.forename: Evan
authors.initials: E
authors.lastname: Adams
authors.affiliation: Pacific Biosciences, Menlo Park, CA, 94025, USA.
authors.forename: Regina
authors.initials: R
authors.lastname: Lam
authors.affiliation: Pacific Biosciences, Menlo Park, CA, 94025, USA.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Turner
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA, 95616, USA.|Faculty of Medicine, Department of Surgery & Cancer, Institute of Reproductive and Developmental Biology, Hammersmith Campus Imperial College London, London, UK.
authors.forename: Erick W
authors.initials: EW
authors.lastname: Loomis
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA, 95616, USA.
authors.forename: Jun Yi
authors.initials: JY
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA, 95616, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hagerman
authors.affiliation: Pacific Biosciences, Menlo Park, CA, 94025, USA. jhanes@pacb.com.
authors.forename: Jeremiah W
authors.identifier: http://orcid.org/0000-0002-2818-6885
authors.initials: JW
authors.lastname: Hanes
mesh_terms: D002460:Cell Line; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D005600:Fragile X Syndrome / Q000235:genetics; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D020080:Tandem Repeat Sequences
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human
keywords: Epigenetic modification; FMR1; Fragile X syndrome; Single molecule sequencing; Tandem repeats; Targeted enrichment
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