Whole‐Genome Amplification of Single‐Cell Genomes for Next‐Generation Sequencing

Christian Korfhage1, Evelyn Fisch1, Evelyn Fricke1, Silke Baedker1, Dirk Loeffert1

1 Qiagen GmbH, Hilden, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.14
DOI:  10.1002/0471142727.mb0714s104
Online Posting Date:  October, 2013
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Abstract

DNA sequence analysis and genotyping of biological samples using next‐generation sequencing (NGS), microarrays, or real‐time PCR is often limited by the small amount of sample available. A single cell contains only one to four copies of the genomic DNA, depending on the organism (haploid or diploid organism) and the cell‐cycle phase. The DNA content of a single cell ranges from a few femtograms in bacteria to picograms in mammalia. In contrast, a deep analysis of the genome currently requires a few hundred nanograms up to micrograms of genomic DNA for library formation necessary for NGS sequencing or labeling protocols (e.g., microarrays). Consequently, accurate whole‐genome amplification (WGA) of single‐cell DNA is required for reliable genetic analysis (e.g., NGS) and is particularly important when genomic DNA is limited. The use of single‐cell WGA has enabled the analysis of genomic heterogeneity of individual cells (e.g., somatic genomic variation in tumor cells). This unit describes how the genome of single cells can be used for WGA for further genomic studies, such as NGS. Recommendations for isolation of single cells are given and common sources of errors are discussed. Curr. Protoc. Mol. Biol. 104:7.14.1‐7.14.11. © 2013 by John Wiley & Sons, Inc.

Keywords: single cell; genome analysis; next‐generation sequencing; somatic genome variation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Single Cells by Dilution
  • Alternate Protocol 1: Preparation of Cell Samples with High Cell Concentration
  • Basic Protocol 2: Cell Lysis, DNA Denaturation, and Whole‐Genome Amplification
  • Support Protocol 1: Cleanup of WGA DNA
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Single Cells by Dilution

  Materials
  • PBS sc (iso‐osmotic cell dilution buffer, free of amplifiable DNA, provided with REPLI‐g Single Cell Kit, Qiagen, cat. no. 150343 or 150345)
  • Cell sample (5000 cells/ml, freshly isolated): eukaryotic cells as well as bacterial cells (Gram‐positive, Gram‐negative) can be used for WGA reactions—plant cells and fungal cells have not been tested so far. (if using plant cells and fungal cells, cell wall digestion may be useful; see unit 2.3)
  • 1.5‐ml reaction tubes

Alternate Protocol 1: Preparation of Cell Samples with High Cell Concentration

  Materials
  • Diluted cell sample ( protocol 1, step 4)
  • REPLI‐g Single Cell Kit (QIAGEN cat.no. 150343 or 150345) containing:
    • Buffer DLB
    • Stop Solution: 1 M DTT
    • H 2O sc
    • REPLI‐g sc Reaction Buffer
    • REPLI‐g sc DNA polymerase
    • 30° and 65°C water baths or thermal cycler

Basic Protocol 2: Cell Lysis, DNA Denaturation, and Whole‐Genome Amplification

  Materials
  • WGA reaction ( protocol 3)
  • 96% to 100% and 70% ethanol
  • TE buffer, pH 8.0 ( appendix 22), autoclaved
  • 45°C water bath or thermal cycler
  • Additional reagents and equipment for spectrophotometric determination of DNA concentration ( appendix 3D or appendix 3J)
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Figures

Videos

Literature Cited

Literature Cited
  Ben‐Ezra, J., Johnson D.A., Rossi, J., Cook, N., and Wu, A. 1991. Effect of fixation on the amplification of nucleic acids from paraffin‐embedded material by the polymerase chain reaction. J. Histochem. Cytochem. 39:351‐354.
  Blanco, L., Bernad, A., Lázaro, J.M., Martín, G., Garmendia, C., and Salas, M. 1989. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase: Symmetrical mode of DNA replication. J. Biol. Chem. 264:8935‐8940.
  Canceill, D., Viguera, E., and Ehrlich, S.D. 1999. Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency. J. Biol. Chem. 274:27481‐27490.
  Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray‐Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, S.F., Egholm, M., and Lasken, R.S. 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. U.S.A. 99:5261‐5266.
  Hosono, S., Faruqi, A.F., Dean, F.B., Du, Y., Zhenyu Sun, Wu, X., Du, J., Kingsmore, S.F., Egholm, M., and Lasken, R.S. 2003. Unbiased whole‐genome amplification directly from clinical samples. Genome Res. 13:954‐964.
  Hou, Y., Song, L., Zhu, P., Zhang, B., Tao, Y., Xu, X., Li, F., Wu, K., Liang, J., Shao, D., Wu, H., Ye, X., Ye, C., Wu, R., Jian, M., Chen, Y., Xie, W., Zhang, R., Chen, L., Liu, X., Yao, X., Zheng, H., Yu, C., Li, Q., Gong, Z., Mao, M., Yang, X., Yang, L., Li, J., Wang, W., Lu, Z., Gu, N., Laurie, G., Bolund, L., Kristiansen, K., Wang, J., Yang, H., Li, Y., Zhang, X., and Wang, J. 2012. Single‐cell exome sequencing and monoclonal evolution of a JAK2‐negative myeloproliferative neoplasm. Cell 148:873‐885.
  Kalisky, T., Blainey, P., and Quake, S.R. 2011. Genomic analysis at the single‐cell level. Annu. Rev. Genet. 45:431‐445.
  Marcy, Y., Ouverney, C., Bik, E.M., Lösekann, T., Ivanova, N., Martin, H.G., Szeto, E., Platt, D., Hugenholtz, P., Relman, D.A., and Quake, S.R. 2007. Dissecting biological “dark matter” with single‐cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc. Natl. Acad. Sci. U.S.A. 104:11889‐11894.
  Melnikov, A., Galinsky, K., Rogov, P., Fennell, T., Van Tyne, D., Russ, C., Daniels, R., Barnes, K.G., Bochicchio, J., Ndiaye, D., Sene, P.D., Wirth, D.F., Nusbaum, C., Volkman, S.K., Birren, B.W., Gnirke, A., and Neafsey, D.E. 2011. Hybrid selection for sequencing pathogen genomes from clinical samples. Genome Biol. 12:R73.
  Navin, N., Krasnitz, A., Rodgers, L., Cook, K., Meth, J., Kendall, J., Riggs, M., Eberling, Y., Troge, J., Grubor, V., Levy, D., Lundin, P., Månér, S., Zetterberg, A., Hicks, J., and Wigler, M. 2010. Inferring tumor progression from genomic heterogeneity. Genome Res. 20:68‐80.
  Odelberg, S.J., Weiss, R.B., Hata, A., and White R. 1995. Template‐switching during DNA synthesis by Thermus aquaticus DNA polymerase. Nucleic Acids Res. 23:2049‐2057.
  Paez, J.G., Lin, L., Beroukhim, R., Lee, J.C., Zhao, X., Richter, D.J., Gabriel, S., Herman, P., Sasaki, H., Altshuler, D., Li, C., Meyerson, M., and Sellers, W.R. 2004. Genome coverage and sequence fidelity of φ29 polymerase‐based multiple strand displacement whole genome amplification. Nucleic Acids Res. 32:e71.
  Quail, M.A., Swerdlow, H., and Turner, D.J. 2009. Improved protocols for the Illumina genome analyzer sequencing system. Curr. Protoc. Hum. Genet. 62:18.2.1‐18.2.27.
  Telenius, H., Carter, N.P., Bebb, C.E., Nordenskjöld, M., Ponder, B.A., and Tunnacliffe, A. 1992. Degenerate oligonucleotide‐primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13:718‐725.
  van Ooyen, S., Loeffert, D., and Korfhage, C. 2011 Overcoming constraints of genomic DNA isolated from paraffin‐embedded tissue. Qiagen GmbH. Available at http://www.qiagen.com/Products/Catalog/Sample‐Technologies/DNA‐Sample‐Technologies/Genomic‐DNA/REPLI‐g‐FFPE‐Kit#resources.
  Viguera, E., Canceill, D., and Ehrlich, S.D. 2001. Replication slippage involves DNA polymerase pausing and dissociation. EMBO J. 20:2587‐2595.
  Weinberg, R. 2007. The Biology of Cancer. Garland Science, New York.
  Zhang, L., Cui, X., Schmitt, K., Hubert, R., Navidi, W., and Arnheim, N. 1992. Whole genome amplification from a single cell: Implications for genetic analysis. Proc. Natl. Acad. Sci. U.S.A. 89:5847‐5851.
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