title: Microarray analysis of copy number variation in single cells.
abstract: We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000-bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5-7 d.
authors.affiliation: Department of Electrical Engineering, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Konings
authors.forename: Evelyne
authors.initials: E
authors.lastname: Vanneste
authors.forename: Sigrun
authors.initials: S
authors.lastname: Jackmaert
authors.forename: Michèle
authors.initials: M
authors.lastname: Ampe
authors.forename: Geert
authors.initials: G
authors.lastname: Verbeke
authors.forename: Yves
authors.initials: Y
authors.lastname: Moreau
authors.forename: Joris Robert
authors.initials: JR
authors.lastname: Vermeesch
authors.forename: Thierry
authors.initials: T
authors.lastname: Voet
mesh_terms: D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D056915:DNA Copy Number Variations; D006801:Humans; D008390:Markov Chains; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide