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.
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We acknowledge all the members of the Hicks and Wigler labs for discussions during the technology development. We thank W. McCombie, E. Ghiban and L. Gelley for their advice and technical assistance, and A. Gordon for informatics support and assistance with figures. N.N. is supported by grants from Texas STARS and the Alice Kleberg Reynolds Foundation. This work was supported by grants to M.W. and J.H. from the Department of the Army (W81XWH04-1-0477), the Breast Cancer Research Foundation, and the Simons Foundation. M.W. is an American Cancer Society Research Professor.
The authors declare no competing financial interests.
Outline of the informatics section. Provide a concise outline of all the steps with input-program-output labels. (EPS 1481 kb)
Input/output data for the informatics procedure of the protocol required to reproduce copy number profiles from single cell sequencing data. Supplementary Data Legend outlines all the data with descriptions. (ZIP 78479 kb)
Contains all programs required to process single cell sequencing data for copy number determination, from genome preparation to sequence mapping and copy number estimation. Supplementary Methods Legend outlines all the programs and their utilities. (ZIP 107 kb)
Descriptions of data and program files. Text providing a brief overview of the data and program files included in the Supplementary Methods and Supplementary Data. (TXT 2 kb)
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Baslan, T., Kendall, J., Rodgers, L. et al. Genome-wide copy number analysis of single cells. Nat Protoc 7, 1024–1041 (2012). https://doi.org/10.1038/nprot.2012.039
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