Single‐Cell Genome and Transcriptome Sequencing Library Construction Using Combination of MDA and Nextera Library Prep Method

Ioanna Andreou1, Christian Korfhage1, Nan Fang1

1 QIAGEN, Hilden
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.23
DOI:  10.1002/0471142727.mb0723s113
Online Posting Date:  January, 2016
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Abstract

Single‐cell analysis gives insights into the heterogeneity of neighboring cells within tissues or within cell populations and is increasing in importance in life science and medicine. Genome and transcriptome sequencing require orders of magnitude of more starting material than what is found in an individual cell. Handling such small quantities means that degradation, sample loss, and contamination can have a pronounced effect on sequence quality and robustness. Recent technical advances in amplification have helped mitigate these challenges. Single‐cell sequencing addresses studies of rare cell types, heterogeneous samples, phenotypes associated with mosaicism or variability, and microbes that cannot be cultured. Single‐cell sequencing can enable the discovery of clonal mutations, cryptic cell types, or transcriptional features that would be diluted or averaged out in bulk tissue. This unit describes the entire workflow from cells to next‐generation sequencing (NGS), including cell lysis, MDA‐based whole‐genome and whole‐transcriptome amplification, and NGS library preparation. © 2016 by John Wiley & Sons, Inc.

Keywords: single cell; whole‐genome amplification; whole‐transcriptome amplification; next‐generation sequencing (NGS); NGS‐library

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Whole‐Genome Amplification Using the REPLI‐g Single Cell Kit
  • Support Protocol 1: PicoGreen Quantification of REPLI‐g‐Amplified DNA
  • Basic Protocol 2: Whole‐Transcriptome Amplification Using the REPLI‐g WTA Single Cell Kit
  • Basic Protocol 3: NGS Library Generation Using a Transposon‐Based Technology
  • Support Protocol 2: Library Quantification Using Real‐Time PCR GeneRead Library Quant Kit for Illumina (Formats A, C, D, E, F, G)
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Whole‐Genome Amplification Using the REPLI‐g Single Cell Kit

  Materials
  • REPLI‐g Single Cell Kit (QIAGEN, cat no. 150343) containing:
    • REPLI‐g single cell (sc) DNA polymerase (blue lid)
    • REPLI‐g single cell (sc) reaction buffer (yellow lid)
    • Buffer DLB (clear lid)
    • Stop solution (red lid)
    • 1× PBS single cell (sc) (clear lid)
    • 1 M DTT (lilac lid)
    • Water single cell (sc)
    • Isolated cells in <4 μl PBS single cell (PBS single cell: PBS buffer specially treated to eliminate any DNA contamination)
  • Thermal cycler
  • Microcentrifuge tubes
  • Microcentrifuge
  • Vortexer

Support Protocol 1: PicoGreen Quantification of REPLI‐g‐Amplified DNA

  Materials
  • Quant‐iT PicoGreen dsDNA reagent (Invitrogen, cat. no. P7581)
  • TE buffer (10 mM Tris·Cl and 1 mM EDTA, pH 8.0)
  • Human genomic DNA (e.g., Promega, cat. no. G3041)
  • REPLI‐g amplified DNA sample (see protocol 1)
  • 2‐ml microcentrifuge tube
  • Aluminum foil
  • 96‐well plates (suitable for use in a fluorescence microplate reader)
  • Fluorescence microplate reader (e.g., TECAN Ultra)
CAUTION: When working with hazardous chemicals, always wear a suitable laboratory coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheet (SDS) available from the product supplier.

Basic Protocol 2: Whole‐Transcriptome Amplification Using the REPLI‐g WTA Single Cell Kit

  Materials
  • REPLI‐g WTA Single Cell Kit containing:
    • Lysis buffer (clear lid)
    • NA denaturation buffer (clear lid)
    • Enzymatic template preparation
    • gDNA Wipeout buffer, WTA (red lid)
    • RT/polymerase buffer (red lid)
    • Random primer (red lid)
    • Oligo dT primer (red lid)
    • Quantiscript RT enzyme mix (red lid)
    • Ligase mix (blue lid)
    • Ligase buffer (blue lid)
    • Amplification of cDNA
    • REPLI‐g sc reaction buffer (yellow lid)
    • REPLI‐g SensiPhi DNA polymerase (yellow lid)
    • H 2O sc
  • Isolated cells in <7 μl PBS sc (PBS single cell)
  • Nuclease‐free water or TE buffer (10 mM Tris·Cl and 1 mM EDTA, pH 8.0)
  • Thermal cycler
  • Microcentrifuge tubes or PCR strips
  • Water bath or heating block
  • Microcentrifuge
  • Vortexer
  • Pipets and pipet tips
  • PCR tubes or plates

Basic Protocol 3: NGS Library Generation Using a Transposon‐Based Technology

  Materials
  • Nextera DNA Sample Preparation Kit (Illumina, cat. no. FC‐121‐1030) containing:
    • Tagment DNA buffer (TD)
    • Tagment DNA enzyme (TDE1)
    • Nextera PCR master mix (NPM)
    • PCR primer cocktail (PPC)
    • Resuspension buffer (RSB)
  • Whole‐genome‐amplified DNA (WGA DNA) or whole‐transcriptome‐amplified cDNA (WTA cDNA)
  • Absolute ethanol (200 proof) (Sigma Aldrich, cat. no. E7023)
  • MinElute PCR Purification Kit (QIAGEN, cat. no. 28004) containing:
    • MinElute spin columns (50, 250)
    • Buffer PB
    • Buffer PE (concentrate)
    • Buffer EB
    • pH Indicator
    • Collection tubes (2‐ml)
    • Loading dye
  • Nextera DNA Sample Preparation Index Kit (24 indices, 96 samples) containing:
    • Buffer RSB
    • Four tubes index primers, N501 to N504
    • Six tubes index primers, N701 to N706
  • Agencourt AMPure XP 60 ml kit (Beckman Coulter Genomics, cat. no. A63881)
  • PCR‐grade water (for gel‐free method)
  • Microcentrifuge, microplate centrifuge
  • Microseal 96‐well PCR plates (Bio‐Rad, cat. no. HSP‐9601) or 0.2‐ml eight‐tube strips and domed cup strips (Bio‐Rad, cat. no. TBS‐0802)
  • Microseal A film (BioRad, cat. no. MSA‐5001)
  • Microseal B adhesive seals (BioRad, cat. no. MSB‐1001)
  • Thermal cycler with heated lid
  • 1.5‐ and 1.7‐ml microcentrifuge tubes
  • TruSeq index plate fixture (labeled rack available from Illumina)
  • 96‐well storage plates, round‐well, 0.8 ml (MIDI plate; Fisher Scientific)
  • RNase/DNase‐free multichannel reagent reservoirs, disposable (VWR, cat. no. 89094‐658)
  • High‐speed microplate shaker
  • Vortexer
  • Magnetic stand
  • Agilent Technologies 2100 BioAnalyzer

Support Protocol 2: Library Quantification Using Real‐Time PCR GeneRead Library Quant Kit for Illumina (Formats A, C, D, E, F, G)

  Materials
  • GeneRead Library Quant Kit (QIAGEN, cat. no. 180612) containing:
    • Laboratory‐verified forward and reverse primers for 500 × 25 μl reactions
    • DNA standard
    • Dilution buffer
    • GeneRead qPCR SYBR Green Mastermix
  • Sample library
  • Molecular‐biology‐grade RNase‐ and DNase‐free water
  • RNase‐/DNase‐free 200‐μl regular PCR tubes (8‐tube or 12‐tube strip) or plates suitable for specific thermal cycler
  • Calibrated single‐ and multi‐channel pipets
  • RNase‐/DNase‐free pipet tips and tubes
  • Centrifuge
  • Optical thin‐wall 8‐cap strips (formats A and D) or optical adhesive film (formats C, E, F, and G)
  • Real‐time PCR instrument
NOTE: Ensure that the GeneRead qPCR SYBR Green Mastermix and the plate format are suitable for the real‐time thermal cycler being utilized.
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Figures

Videos

Literature Cited

Literature Cited
  Adey, A., Morrison, H.G., Asan, Xun, X., Kitzman, J.O., Turner, E.H., Stackhouse, B., MacKenzie, A.P., Caruccio, N.C., Zhang, X., and Shendure, J. 2010. Rapid, low‐input, low‐bias construction of shotgun fragment libraries by high‐density in vitro transposition. Genome Biol. 11:R119. doi: 10.1186/gb-2010-11-12-r119.
  de Bourcy, C.F., De Vlaminck, I., Kanbar, J.N., Wang, J., Gawad, C., and Quake, S.R. 2014. A quantitative comparison of single‐cell whole genome amplification methods. PloS One 9:e105585. doi: 10.1371/journal.pone.0105585.
  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. doi: 10.1073/pnas.082089499.
  Frohlich, J. and Konig, H. 2000. New techniques for isolation of single prokaryotic cells. FEMS Microbiol. Rev. 24:567‐572. doi: 10.1111/j.1574-6976.2000.tb00558.x.
  Lamble, S., Batty, E., Attar, M., Buck, D., Bowden, R., Lunter, G., Crook, D., El‐Fahmawi, B., and Piazza, P. 2013. Improved workflows for high throughput library preparation using the transposome‐based Nextera system. BMC Biotechnol. 13:104‐114. doi: 10.1186/1472-6750-13-104.
  Lazinski, D.W. and Camilli, A. 2013. Homopolymer tail‐mediated ligation PCR: A streamlined and highly efficient method for DNA cloning and library construction. BioTechniques 54:25‐34. doi: 10.2144/000113981.
  Marine, R., Polson, S.W., Ravel, J., Hatfull, G., Russell, D., Sullivan, M., Syed, F., Dumas, M., and Wommack, K.E. 2011. Evaluation of a transposase protocol for rapid generation of shotgun high‐throughput sequencing libraries from nanogram quantities of DNA. Appl. Environ. Microbiol. 77:8071‐8079. doi: 10.1128/AEM.05610-11.
  Navin, N. and Hicks, J. 2011. Future medical applications of single‐cell sequencing in cancer. Genome Med. 3:31‐43. doi: 10.1186/gm247.
  Nawy, T. 2014. Single‐cell sequencing. Nat. Methods 11:18. doi: 10.1038/nmeth.2771.
  Oyola, S.O., Otto, T.D., Gu, Y., Maslen, G., Manske, M., Campino, S., Turner, D.J., Macinnis, B., Kwiatkowski, D.P., Swerdlow, H.P., and Quail, M.A. 2012. Optimizing Illumina next‐generation sequencing library preparation for extremely AT‐biased genomes. BMC Genomics 13:1. doi: 10.1186/1471-2164-13-1.
  Piggot, P.J. and Coote, J.G. 1976. Genetic aspects of bacterial endospore formation. Bacteriol. Rev. 40:908‐962.
  Raghunathan, A., Ferguson, H.R., Jr., Bornarth, C.J., Song, W., Driscoll, M., and Lasken, R.S. 2005. Genomic DNA amplification from a single bacterium. Appl. Environ. Microbiol. 71:3342‐3347. doi: 10.1128/AEM.71.6.3342-3347.2005.
  Syed, F., Grunenwald, H., and Caruccio, N. 2009. Next‐generation sequencing library preparation: Simultaneous fragmentation and tagging using in vitro transposition. Nat. Methods 6: Application Notes ISSN: 1548‐7091 EISSN: 1548‐7105.
Internet Resources
  http://www.qiagen.com/de/products/catalog/sample‐technologies/dna‐sample‐technologies/genomic‐dna/repli‐g‐single‐cell‐kit/
  Link to RepliG Kits for MDA‐based WGA and WTA.
  https://tools.lifetechnologies.com/content/sfs/manuals/mp32850.pdf
  Link to products for Qubit‐based DNA quantification.
  http://support.illumina.com/content/dam/illumina‐support/documents/myillumina/2a3297c5‐8a34‐4fc5‐a148‐3e16666fd65e/nextera_dna_sample_prep_guide_15027987_b.pdf
  Dual Indexing and Low Plexity Pooling Guidelines
  http://www.qiagen.com/de/resources/resourcedetail?id=600ea7b8‐93b0‐444a‐b098‐968cddc113ab&lang=en
  Link to Illumina's guidelines for choosing the right adaptors.
  http://www.sabiosciences.com/genereaddataanalysis.php
  Link to Illumina's user guide for Nextera‐based library construction.
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