Parallel WGA and WTA for Comparative Genome and Transcriptome NGS Analysis Using Tiny Cell Numbers

Christian Korfhage1, Evelyn Fricke1, Andreas Meier1

1 Qiagen, Hilden
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.19
DOI:  10.1002/0471142727.mb0719s111
Online Posting Date:  July, 2015
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Abstract

Genomic DNA determines how and when the transcriptome is changed by a trigger or environmental change and how cellular metabolism is influenced. Comparative genome and transcriptome analysis of the same cell sample links a defined genome with all changes in the bases, structure, or numbers of the transcriptome. However, comparative genome and transcriptome analysis using next‐generation sequencing (NGS) or real‐time PCR is often limited by the small amount of sample available. In mammals, the amount of DNA and RNA in a single cell is ∼10 picograms, but deep analysis of the genome and transcriptome currently requires several hundred nanograms of nucleic acids for library preparation for NGS sequencing. Consequently, accurate whole‐genome amplification (WGA) and whole‐transcriptome amplification (WTA) is required for such quantitative analysis. This unit describes how the genome and the transcriptome of a tiny number of cells can be amplified in a highly parallel and comparable process. Protocols for quality control of amplified DNA and application of amplified DNA for NGS are included. © 2015 by John Wiley & Sons, Inc.

Keywords: genome analysis; transcriptome analysis; cancer; next‐generation sequencing (NGS); somatic genome variation; whole‐genome amplification (WGA); whole‐transcriptome amplification (WTA)

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Lysed Cell Samples for Parallel WGA and WTA
  • Alternate Protocol 1: Preparation of Purified DNA/RNA Samples for Parallel WGA and WTA
  • Basic Protocol 2: Parallel Whole‐Genome and Whole‐Transcriptome Amplification
  • Support Protocol 1: Quality Control of WTA and WGA DNA by Real‐Time PCR
  • Support Protocol 2: Cleanup of WGA and WTA DNA using Agencourt Ampure XP Magnetic Beads
  • Support Protocol 3: Cleanup of WGA and WTA DNA using LiCl/EtOH Precipitation
  • Basic Protocol 3: Use of WTA and WGA DNA for Next‐Generation Sequencing
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Lysed Cell Samples for Parallel WGA and WTA

  Materials
  • Cell sample (105 to 107 cells/ml in PBS, freshly isolated)
  • Trypan blue counting kit (Bio‐Rad, cat. no. 145‐0003)
  • PBS ( appendix 22), freshly prepared
  • Dry ice
  • Lysis buffer from REPLI‐g Cell WGA & WTA Kit (Qiagen, cat. no. 150052 or 150054)
  • Microcentrifuge tubes
  • Automated cell counter (e.g., Bio‐Rad TC20, cat. no. 145‐0102) with counting slide
  • Thermal cycler or water bath(s)

Alternate Protocol 1: Preparation of Purified DNA/RNA Samples for Parallel WGA and WTA

  Materials
  • Purified nucleic acid solution containing RNA and DNA from the same sample
  • NA denaturation buffer from REPLI‐g Cell WGA & WTA Kit (Qiagen, cat. no. 150052 or 150054)
  • Microcentrifuge tubes
  • Thermal cycler or water bath(s)

Basic Protocol 2: Parallel Whole‐Genome and Whole‐Transcriptome Amplification

  Materials
  • Two 10‐μl aliquots of cell lysate or purified nucleic acids from the same sample (see protocol 1 or protocol 2Alternate Protocol)
  • REPLI‐g Cell WGA & WTA Kit (Qiagen, cat. no. 150052 or 150054) containing:
    • RT polymerase buffer
    • gDNA wipeout buffer
    • H 2O sc
    • Random primers
    • Oligo(dT) primer
    • WGA Ready enzyme
    • Quantiscript RT enzyme mix
    • Ligase buffer
    • Ligase mix
    • REPLI‐g sc reaction buffer
    • REPLI‐g SensiPhi DNA polymerase
  • Thermal cycler or water bath(s)
NOTE: All reaction mixes should be scaled up accordingly when analyzing more than one sample at one time.

Support Protocol 1: Quality Control of WTA and WGA DNA by Real‐Time PCR

  Materials
  • WTA and WGA DNA (see protocol 3)
  • Agencourt AMPure XP Beads (Beckman Coulter, cat. no. A63880)
  • TE buffer, pH 8.0 ( appendix 22)
  • 70% (v/v) ethanol, stored no longer than 7 days
  • Microcentrifuge tubes
  • Magnetic bead concentrator (e.g., Dyna Mag, Life Technologies, cat. no. 123.21)
NOTE: Use ethanol and TE buffers prepared separately (one for WGA DNA and one for WTA DNA) to avoid cross‐contamination.

Support Protocol 2: Cleanup of WGA and WTA DNA using Agencourt Ampure XP Magnetic Beads

  Materials
  • WTA and WGA DNA (see protocol 3)
  • TE buffer, pH 8.0 ( appendix 22)
  • 70% and 96‐100% (v/v) ethanol
  • Lithium chloride (7.5 M)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • Microcentrifuge tubes
NOTE: Use ethanol and TE buffers prepared separately (one for WGA DNA and one for WTA DNA) to avoid cross‐contamination.

Support Protocol 3: Cleanup of WGA and WTA DNA using LiCl/EtOH Precipitation

  Materials
  • Quant‐iT PicoGreen dsDNA reagent (Invitrogen, cat no. P7581) or Qubit dsDNA BR Assay system (Invitrogen, cat. no. Q32850)
  • TE buffer, pH 8 ( appendix 22)
  • Nuclease‐free water
  • MinElute PCR Purification Kit (Qiagen, cat. no. 28004 or 28006), including columns and Buffer EB
  • GeneRead NGS Library I Core Kit (Qiagen, cat. no. 180432)
  • GeneRead Library Quant Kit (Qiagen, cat. no. 180612) or Real‐time PCR Kapa Kit (peqLab, cat. no. 07‐KK4822)
  • GeneRead Adapter I Kit (Qiagen, cat. no. 180912 or 180984)
  • GeneRead DNA I Amp Kit (Qiagen, cat. no 180455)
  • GeneRead Size Selection Kit (Qiagen, cat. no. 180514)
  • Microcentrifuge tubes
  • DNA shearing device (e.g., Covaris S220)
  • Agilent Bioanalyzer, Agilent DNA 7500 chip (cat. no. 5067‐150), and associated reagent set
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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.
  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.
  Cho, R.J., Campbell, M.J., Winzeler, E.A., Steinmetz, L., Conway, A., Wodicka, L., Wolfsberg, T.G., Gabrielian, A.E., Landsman, D., Lockhart, D.J., and Davis, R.W. 1998. A genome‐wide transcriptional analysis of the mitotic cell cycle. Mol. Cell 2:65‐73.
  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.
  Gaglio, D., Soldati, C., Vanoni, M., Alberghina, L., and Chiaradonna, F. 2009. Glutamine deprivation induces abortive S‐phase rescued by deoxyribonucleotides in K‐ras transformed fibroblasts. PLoS One 4:e4715.
  Hosono, S., Faruqi, A.F., Dean, F.B., Du, Y., Zhenyu, S., 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.
  Korfhage, C., Fisch, E., Fricke, E., Baedker, S., and Loeffert, D. 2013. Whole‐genome amplification of single‐cell genomes for next‐generation sequencing. Curr. Protoc. Mol. Biol. 104:7.14.1‐7.14.11.
  Mathews, E.H., Stander, B.A., Joubert, A.M., and Liebenberg, L. 2014. Tumor cell culture survival following glucose and glutamine deprivation at typical physiological concentrations. Nutrition 30:218‐227.
  Mignone, F., Gissi, C., Liuni, S., and Pesole, G. 2002. Untranslated regions of mRNAs. Genome Biol. 3:reviews0004.1‐0004.10.
  Miranda, K.C., Bond, D.T., Levin, J.Z., Adiconis, X., Sivachenko, A., Russ, C., Brown, D., Nusbaum, C., and Russo, L.M. 2014. Massively parallel sequencing of human urinary exosome/microvesicle RNA reveals a predominance of non‐coding RNA. PLoS One 9:e96094.
  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.
  Viguera, E., Canceill, D., and Ehrlich, S.D. 2001. Replication slippage involves DNA polymerase pausing and dissociation. EMBO J. 20:2587‐2595.
  Whitfield, M.L., Sherlock, G., Saldanha, A.J., Murray, J.I., Ball, C.A., Alexander, K.E., Matese, J.C., Perou, C.M., Hurt, M.M., Brown, P.O., and Botstein, D. 2002. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol. Biol. Cell. 13:1977‐2000.
Internet Resource
  http://www.qiagen.com/Products/Catalog/Sample‐Technologies/DNA‐Sample‐Technologies/Genomic‐DNA/REPLI‐g‐FFPE‐Kit#resources
  Tips for overcoming constraints of genomic DNA isolated from paraffin‐embedded tissue.
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