title: Genome-wide copy number analysis of single cells.
abstract: Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ∼3 d from flow cytometry to sequence-ready DNA libraries.
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
authors.forename: Timour
authors.initials: T
authors.lastname: Baslan
authors.forename: Jude
authors.initials: J
authors.lastname: Kendall
authors.forename: Linda
authors.initials: L
authors.lastname: Rodgers
authors.forename: Hilary
authors.initials: H
authors.lastname: Cox
authors.forename: Mike
authors.initials: M
authors.lastname: Riggs
authors.forename: Asya
authors.initials: A
authors.lastname: Stepansky
authors.forename: Jennifer
authors.initials: J
authors.lastname: Troge
authors.forename: Kandasamy
authors.initials: K
authors.lastname: Ravi
authors.forename: Diane
authors.initials: D
authors.lastname: Esposito
authors.forename: B
authors.initials: B
authors.lastname: Lakshmi
authors.forename: Michael
authors.initials: M
authors.lastname: Wigler
authors.forename: Nicholas
authors.initials: N
authors.lastname: Navin
authors.forename: James
authors.initials: J
authors.lastname: Hicks
mesh_terms: D000465:Algorithms; D002467:Cell Nucleus / Q000235:genetics; D056915:DNA Copy Number Variations; D005434:Flow Cytometry; D018740:Genetic Heterogeneity; D005821:Genetic Techniques; D006801:Humans; D059010:Single-Cell Analysis / Q000379:methods*