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Microarray analysis of copy number variation in single cells


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.

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Figure 1
Figure 2: Typical plot of single-cell WGA BAC array results after mixture model analysis.
Figure 3: Typical reference model for single-cell WGA BAC array data analysis.
Figure 4: SNP array results.
Figure 5: Graphical presentation of the intersection of the various models.

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We are grateful to C. Melotte and P. Brady for the critical reading of the manuscript, to the Mapping Core and Map finishing groups of the Wellcome Trust Sanger Institute for initial BAC clone supply and verification, as well as to the microarray facility of the Flanders Interuniversity Institute for Biotechnology (VIB) for their help in spotting the arrays. This work was made possible by grants from the IWT (SBO-60848 to J.R.V. and Y.M.; TBM-090878 to J.R.V., T.V. and Y.M.); FWO-G.A093.11 to T.V. and J.R.V.; KUL PFV/10/016 SymBioSys to Y.M., J.R.V. and T.V.; GOA/12/015 to J.R.V.; and KUL GOA MaNet and EU FP7-Health CHeartED grants to Y.M. E.V. was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

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Authors and Affiliations



P.K., M.A., G.V., Y.M. designed the algorithms to analyze and combine BAC and SNP array results. E.V., T.V. and J.R.V. have developed the methods to analyze single cells using SNP-arrays. J.R.V., E.V., S.J. and T.V. developed the wet-lab protocols. E.V., T.V. and P.K. wrote the manuscript and all authors reviewed the manuscript. Y.M., J.R.V. and T.V. obtained the funding.

Corresponding authors

Correspondence to Joris Robert Vermeesch or Thierry Voet.

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The authors declare no competing financial interests.

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Supplementary Data

Setup of Affymetrix reference data. (DOC 28 kb)

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Konings, P., Vanneste, E., Jackmaert, S. et al. Microarray analysis of copy number variation in single cells. Nat Protoc 7, 281–310 (2012).

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