Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Microarray analysis of copy number variation in single cells

Abstract

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.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Vanneste, E. et al. Chromosome instability is common in human cleavage-stage embryos. Nat. Med. 15, 577–583 (2009).

    CAS  Article  Google Scholar 

  2. Vanneste, E. et al. Genome-wide single cell array analysis for preimplantation genetic diagnosis of a complex chromosomal rearrangement carrier. Hum. Reprod. 18 (Suppl 1): 27 (2010).

    Google Scholar 

  3. Spits, C. et al. Whole-genome multiple displacement amplification from single cells. Nat. Protoc. 1, 1965–1970 (2006).

    CAS  Article  Google Scholar 

  4. Treff, N.R., Su, J., Tao, X., Levy, B. & Scott, R.T. Accurate single cell 24 chromosome aneuploidy screening using whole genome amplification and single nucleotide polymorphism microarrays. Fertil. Steril. 94, 2017–2021 (2010).

    CAS  Article  Google Scholar 

  5. Johnson, D.S. et al. Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol. Hum. Reprod. 25, 1066–1075 (2010).

    CAS  Article  Google Scholar 

  6. Alfarawati, S., Fragouli, E., Colls, P. & Wells, D. First births after preimplantation genetic diagnosis of structural chromosome abnormalities using comparative genomic hybridization and microarray analysis. Hum. Reprod. 26, 1560–1574 (2011).

    CAS  Article  Google Scholar 

  7. Hu, D.G., Webb, G. & Hussey, N. Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization. Mol. Hum. Reprod. 4, 283–289 (2004).

    Article  Google Scholar 

  8. Le Caignec, C. et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res. 34, e68 (2006).

    Article  Google Scholar 

  9. Hellani, A., Abu-Amero, K., Azouri, J. & El-Akoum, S. Successful pregnancies after application of array-comparative genomic hybridization in PGS-aneuploidy screening. Reprod. Biomed. Online 17, 841–847 (2008).

    CAS  Article  Google Scholar 

  10. Fiegler, H. et al. High resolution array-cgh analysis of single cells. Nucleic Acids Res. 35, e15 (2007).

    Article  Google Scholar 

  11. Geigl, J.B. et al. Identification of small gains and losses in single cells after whole genome amplification on tiling oligo arrays. Nucleic Acids Res. 37, e105 (2009).

    Article  Google Scholar 

  12. Gutierrez-Mateo, C. et al. Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos. Fertil. Steril. 95, 953–958 (2011).

    CAS  Article  Google Scholar 

  13. Fiorentino, F. et al. PGD for reciprocal and Robertsonian translocations using array comparative genomic hybridization. Hum. Reprod. 26, 1925–1935 (2011).

    CAS  Article  Google Scholar 

  14. Iwamoto, K. et al. Detection of chromosomal structural alterations in single cells by SNP arrays: a systematic survey of amplification bias and optimized workflow. PLoS ONE 2, e1306 (2007).

    Article  Google Scholar 

  15. Treff, N.R., Su, J., Tao, X., Northrop, L.E. & Scott, R.T. Single cell whole genome amplification technique impacts the accuracy of SNP microarray based genotyping and copy number analysis. Mol. Hum. Reprod. 17, 335–343 (2011).

    CAS  Article  Google Scholar 

  16. Handyside, A.H. et al. Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes. J. Med. Gen. 47, 651–658 (2009).

    Article  Google Scholar 

  17. Ampe, M., Verbeke, G., Vanneste, E. & Vermeesch, J.R. Analysis of array CGH data for the detection of single-cell chromosomal imbalances. Online J. Bioinformatics 11, 224–244 (2010).

    Google Scholar 

  18. Voet, T et al. Breakage-fusion-bridge cycles leading to inv dup del occur in human cleavage stage embryos. Hum. Mutat. 32, 783–793 (2011).

    CAS  Article  Google Scholar 

  19. Di, X. et al. Dynamic model based algorithms for screening and genotyping over 100 K SNPs on oligonucleotide microarrays. Bioinformatics 21, 1958–1963 (2005).

    CAS  Article  Google Scholar 

  20. Nannya, Y. et al. A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays. Cancer Res. 65, 6071–6079 (2005).

    CAS  Article  Google Scholar 

  21. Yurov, Y.B., Vorsanova, S.G. & Iourov, I.Y. GIN'n'CIN hypothesis of brain aging: deciphering the role of somatic genetic instabilities and neural aneuploidy during ontogeny. Mol. Cytogenet. 2, 23 (2009).

    Article  Google Scholar 

  22. Stratton, M.R., Campbell, P.J. & Futreal, P.A. The cancer genome. Nature 458, 719–724 (2009).

    CAS  Article  Google Scholar 

  23. Mkrtchyan, H. et al. Early embryonic chromosome instability results in stable mosaic pattern in human tissues. PLoS ONE 5, e9591 (2010).

    Article  Google Scholar 

  24. Conlin, L.K. et al. Mechanisms of mosaicism, chimerism and uniparental disomy identified by single nucleotide polymorphism array analysis. Hum. Mol. Genet. 19, 1263–1275 (2010).

    CAS  Article  Google Scholar 

  25. Coufal, N. et al. L1 retrotransposition in human neural progenitor cells. Nature 460, 1127–1131 (2009).

    CAS  Article  Google Scholar 

  26. Piotrowski, A. et al. Somatic mosaicism for copy number variation in differentiated human tissues. Hum. Mutat. 29, 1118–1124 (2008).

    Article  Google Scholar 

  27. Ballif, B.C. et al. Detecting sex chromosome anomalies and common triploidies in products of conception by array-based comparative genomic hybridization. Prenat. Diagn. 26, 333–339 (2006).

    CAS  Article  Google Scholar 

  28. McLachlan, G. & Peel, D. Finite Mixture Models (Wiley-Interscience, 2000).

  29. Böhning, D. Computer Assisted Analysis of Mixtures and Applications: Meta-Analysis, Disease Mapping, and Others (Chapman & Hall/CRC, 2000).

  30. Durbin, R., Eddy, S., Krogh, A. & Mitchison, G. Biological Sequence Analysis (Cambridge University Press, 1998).

Download references

Acknowledgements

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).

Author information

Authors and Affiliations

Authors

Contributions

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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data

Setup of Affymetrix reference data. (DOC 28 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Konings, P., Vanneste, E., Jackmaert, S. et al. Microarray analysis of copy number variation in single cells. Nat Protoc 7, 281–310 (2012). https://doi.org/10.1038/nprot.2011.426

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.426

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing