Genome-wide copy number analysis of single cells

• Published in: Nature protocols Vol. 7; no. 6; pp. 1024 - 1041
• Main Authors: Baslan, Timour, Kendall, Jude, Rodgers, Linda, Cox, Hilary, Riggs, Mike, Stepansky, Asya, Troge, Jennifer, Ravi, Kandasamy, Esposito, Diane, Lakshmi, B, Wigler, Michael, Navin, Nicholas, Hicks, James
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.06.2012; Nature Publishing Group
• Subjects: 631/1647/2217/2220; 631/1647/2217/457/649/2157; 631/1647/48; 631/1647/666/2261; Admixtures; Algorithms; Analytical Chemistry; Bar codes; Biological Techniques; Biology; Cancer; Cell Nucleus - genetics; Computational Biology/Bioinformatics; Copy number; Copy number variations; Deoxyribonucleic acid; DNA; DNA Copy Number Variations; DNA sequencing; Flow Cytometry; Genetic Heterogeneity; Genetic screening; Genetic Techniques; Genomes; genomics; Heterogeneity; Humans; Identification and classification; Informatics; Laboratories; Life Sciences; Medical research; Metastasis; Methods; Microarrays; Multiplexing; Next-generation sequencing; Nuclei; Nuclei (cytology); Nucleotide sequence; Organic Chemistry; Phenotypic variations; protocol; Single-Cell Analysis - methods; Tumors; Canada; New York; United States > US; Texas
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2012.039

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.