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Copy Number Variation Analysis by Droplet Digital PCR

  • Suvi K. Härmälä
  • Robert Butcher
  • Chrissy H. Roberts
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1654)

Abstract

The health impact of many copy number variants in our genome remains still largely to be discovered. Detecting and genotyping this often complex variation presents a technical challenge. Here we describe a 96-well format droplet digital PCR (ddPCR) protocol for genotyping a common copy variant in the human haptoglobin gene. ddPCR allows for high-throughput and accurate quantitation of gene copy numbers.

Key words

Droplet digital PCR ddPCR Copy number variation CNV Genotyping Haptoglobin HP 

Notes

Acknowledgments

This work was supported by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement (MC-A760-5QX00). Further funding was provided by the UK Biotechnology and Biological Sciences Research Council (BBSRC BB/M009513/1 to SKH). We thank BJ Hennig for access MRC Keneba Biobank (The Gambia) samples and data, and special thanks to all participants and staff at MRC Keneba, The Gambia. RB is supported by the Wellcome Trust (098521/B/12/Z). ChR is supported by the Wellcome Trust Institutional Strategic Support Fund (105609/Z/14/Z).

References

  1. 1.
    Henrichsen CN, Chaignat E, Reymond A (2009) Copy number variants, diseases and gene expression. Hum Mol Genet 18:1–8CrossRefGoogle Scholar
  2. 2.
    Buchanan JA, Scherer SW (2008) Contemplating effects of genomic structural variation. Genet Med 10:639–647CrossRefPubMedGoogle Scholar
  3. 3.
    Jiang W, Johnson C, Jayaraman J et al (2012) Copy number variation leads to considerable diversity for B but not a haplotypes of the human KIR genes encoding NK cell receptors. Genome Res 22:1845–1854CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Weaver S, Dube S, Mir A et al (2010) Taking qPCR to a higher level: analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution. Methods 50:271–276CrossRefPubMedGoogle Scholar
  5. 5.
    D’haene B, Vandesompele J, Hellemans J (2010) Accurate and objective copy number profiling using real-time quantitative PCR. Methods 50:262–270CrossRefPubMedGoogle Scholar
  6. 6.
    Rose-Zerilli MJ, Barton SJ, Henderson AJ et al (2009) Copy-number variation genotyping of GSTT1 and GSTM1 gene deletions by real-time PCR. Clin Chem 55:1680–1685CrossRefPubMedGoogle Scholar
  7. 7.
    Hindson BJ, Ness KD, Masquelier DA et al (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83:8604–8610CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Pinheiro LB, Coleman VA, Hindson CM et al (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84:1003–1011CrossRefPubMedGoogle Scholar
  9. 9.
    Hindson CM, Chevillet JR, Briggs HA et al (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10(10):1003–1005. doi: 10.1038/nmeth.2633 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Roberts CH, Jiang W, Jayaraman J et al (2014) Killer-cell immunoglobulin-like receptor gene linkage and copy number variation analysis by droplet digital PCR. Genome Med 6:20CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Langlois MR, Delanghe JR (1996) Biological and clinical significance of haptoglobin polymorphism in humans. Clin Chem 42:1589–1600PubMedGoogle Scholar
  12. 12.
    Maeda N, Yang F, Barnett DR et al (1984) Duplication within the haptoglobin Hp2 gene. Nature 309:131–135CrossRefPubMedGoogle Scholar
  13. 13.
    Kristiansen M, Graversen JH, Jacobsen C et al (2001) Identification of the haemoglobin scavenger receptor. Nature 409:198–201CrossRefPubMedGoogle Scholar
  14. 14.
    Nielsen MJ, Moestrup SK (2009) Receptor targeting of hemoglobin mediated by the haptoglobins: roles beyond heme scavenging. Blood 114:764–771CrossRefPubMedGoogle Scholar
  15. 15.
    Okazaki T, Yanagisawa Y, Nagai T (1997) Analysis of the affinity of each haptoglobin polymer for hemoglobin by two-dimensional affinity electrophoresis. Clin Chim Acta 258:137–144CrossRefPubMedGoogle Scholar
  16. 16.
    Wejman JC, Hovsepian D, Wall JS et al (1984) Structure and assembly of Haptoglobin polymers by electron microscopy. J Mol Biol 174:343–368CrossRefPubMedGoogle Scholar
  17. 17.
    R Core Team (2015) R: a language and environment for statistical computing. In: R Found. Stat. Comput. https://www.r-project.org/. Accessed 31 Jul 2016
  18. 18.
    Rozen S, Skaletzky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols (methods in molecular biology). Humana Press, Totowa, NJ, pp 365–386Google Scholar
  19. 19.
    Jullien N (2013) AmplifX 1.7.0. http://crn2m.univ-mrs.fr/pub/amplifx-dist. Accessed 31 Jul 2016
  20. 20.
    National Centre for Biotechnology Information (2016) Variation Viewer http://www.ncbi.nlm.nih.gov/variation/view/. Accessed 31 Jul 2016
  21. 21.
    Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  22. 22.
    Luo W, Yang H, Rathbun K et al (2005) Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR Assay. J Clin Microbiol 43:1851–1857CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Bio-Rad (2014) Droplet digital PCR applications guide. pp. 1–111. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf/. Accessed 5 July 2017
  24. 24.
    Karlin-Neumann G, Montesclaros L, Heredia N et al (2012) Probing copy number variations using bio-Rad’s QX100™ droplet digital™ PCR system. BioRad Tech Bull 6277. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/bulletin_6277.pdf. Accessed 5 July 2017
  25. 25.
    Dingle TC, Hall Sedlak R, Cook L, Jerome KR (2013) Tolerance of droplet-digital PCR versus real-time quantitative PCR to inhibitory substances. Clin Chem 59:1670–1672CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Suvi K. Härmälä
    • 1
  • Robert Butcher
    • 2
  • Chrissy H. Roberts
    • 2
  1. 1.MRC International Nutrition GroupLondon School of Hygiene & Tropical MedicineLondonUK
  2. 2.Department of Clinical ResearchLondon School of Hygiene & Tropical MedicineLondonUK

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