title: A Torrent of data: mapping chromatin organization using 5C and high-throughput sequencing.
abstract: The study of three-dimensional genome organization is an exciting research area, which has benefited from the rapid development of high-resolution molecular mapping techniques over the past decade. These methods are derived from the chromosome conformation capture (3C) technique and are each aimed at improving some aspect of 3C. All 3C technologies use formaldehyde fixation and proximity-based ligation to capture chromatin contacts in cell populations and consider in vivo spatial proximity more or less inversely proportional to the frequency of measured interactions. The 3C-carbon copy (5C) method is among the most quantitative of these approaches. 5C is extremely robust and can be used to study chromatin organization at various scales. Here, we present a modified 5C analysis protocol adapted for sequencing with an Ion Torrent Personal Genome Machine™ (PGM™). We explain how Torrent 5C libraries are produced and sequenced. We also describe the statistical and computational methods we developed to normalize and analyze raw Torrent 5C sequence data. The Torrent 5C protocol should facilitate the study of in vivo chromatin architecture at high resolution because it benefits from high accuracy, greater speed, low running costs, and the flexibility of in-house next-generation sequencing.
authors.affiliation: Department of Biochemistry and Goodman Cancer Research Center, McGill University, Montréal, Québec, Canada.
authors.forename: James
authors.initials: J
authors.lastname: Fraser
authors.forename: Sylvain D
authors.initials: SD
authors.lastname: Ethier
authors.forename: Hisashi
authors.initials: H
authors.lastname: Miura
authors.forename: Josée
authors.initials: J
authors.lastname: Dostie
mesh_terms: D002454:Cell Differentiation; D002843:Chromatin / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D002874:Chromosome Mapping / Q000295:instrumentation / Q000379:methods; D019295:Computational Biology / Q000379:methods; D005557:Formaldehyde / Q000737:chemistry; D015723:Gene Library; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D018398:Homeodomain Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009690:Nucleic Acid Conformation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013223:Statistics as Topic / Q000379:methods*; D013997:Time Factors
chemical_list: D002843:Chromatin; D018398:Homeodomain Proteins; C088144:HoxA protein; D005557:Formaldehyde