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Genome-wide copy number analysis of single cells

A Corrigendum to this article was published on 25 February 2016

This article has been updated

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.

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Figure 1: Schematic of the experimental workflow of SNS.
Figure 2: Flow sorting of single nuclei on the basis of DNA content.
Figure 3: WGA amplification profiles of single-cell DNA from four different single cells.
Figure 4: Sonication profiles of WGA DNA using different sonication programs on the Covaris E210 instrument.
Figure 5: Sequencing library size profiles following AMPure bead purification using different volumes of beads.
Figure 6: Schematic of the informatics workflow of SNS.
Figure 7: Genome plot of normalized bin counts and segmentation illustrating the genome-wide copy number profile of the single-cell example SRR054616.
Figure 8: Density plot of segment value differences.
Figure 9: A close-up view of a region on chromosome 4 illustrating normalized bin count for each bin on the chromosomal segment.
Figure 10: Representative illustration of genome sector loss (GSL), where large homozygous deletions and patterns consistent with chromosomal 'shredding' are evident.

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  • 24 February 2016

     In the version of this article initially published, the units for the concentration of NaCl in the NST buffer described in the Reagent Setup section were incorrect. The correct unit should be mM. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

T.B., J.K., N.N., J.H. and M.W. designed methods and analyzed data. T.B., L.R., H.C., M.R., A.S. and J.T. performed experiments. K.R., D.E. and B.L. contributed to the technology development. J.K., N.N., T.B., J.H. and M.W. developed informatic analysis. T.B., J.K. and J.H. wrote the paper.

Corresponding author

Correspondence to James Hicks.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Outline of the informatics section. Provide a concise outline of all the steps with input-program-output labels. (EPS 1481 kb)

Supplementary Data

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)

Supplementary Methods

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)

Supplementary Note

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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