Single Molecule Sequencing with a HeliScope Genetic Analysis System

John F. Thompson1, Kathleen E. Steinmann1

1 Helicos BioSciences, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.10
DOI:  10.1002/0471142727.mb0710s92
Online Posting Date:  October, 2010
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Abstract

Helicos™ Single Molecule Sequencing (SMS) provides a unique view of genome biology through direct sequencing of cellular nucleic acids in an unbiased manner, providing both accurate quantitation and sequence information. Sample preparation does not require ligation or PCR amplification, avoiding the GC‐content and size biases observed in other technologies. DNA is simply sheared, tailed with poly(A), and hybridized to a flow cell surface containing oligo(dT) for sequencing‐by‐synthesis of billions of molecules in parallel. This process also requires far less material than other technologies. Gene expression measurements can be done using first‐strand cDNA‐based methods (RNA‐Seq) or using a novel approach that allows direct hybridization and sequencing of cellular RNA for the most direct quantitation possible. In this unit, principles and methods for using the Helicos® Genetic Analysis System are discussed. Curr. Protoc. Mol. Biol. 92:7.10.1‐7.10.14. © 2010 by John Wiley & Sons, Inc.

Keywords: Next Gen Sequencing; DNA shearing; FFPE sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Shearing and Purification of DNA in Preparation for Tailing
  • Basic Protocol 2: dA‐Tailing of DNA Molecules by Terminal Transferase
  • Basic Protocol 3: DNA Sequencing Using the HeliScope™ Genetic Analysis System
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Shearing and Purification of DNA in Preparation for Tailing

  Materials
  • DNA of interest (see annotations to step 5)
  • 10× TE buffer, pH 8.0 (Invitrogen)
  • Agencourt AMPure XP Kit (Agencourt Bioscience)
  • 70% ethanol (prepare fresh by mixing 7 ml absolute ethanol and 3 ml distilled H 2O; do not use stock 70% ethanol as the ethanol concentration changes with time)
  • S2 instrument (Covaris); for higher‐throughput applications, the E210 instrument may be used
  • MicroTube holder (single tube; Covaris)
  • Preparation Station (Covaris)
  • Snap‐cap microTubes with AFA fiber and pre‐split Teflon/silicone/Teflon septa (Covaris)
  • Tabletop centrifuge with appropriate adaptors
  • 1.5‐ml MAXYMum Recovery tubes (Axygen Scientific)
  • Dynal Magnet: DynaMag ‐2 Magnet (Invitrogen) or equivalent
  • Heat block milled for 1.5‐ml tubes (VWR Scientific)

Basic Protocol 2: dA‐Tailing of DNA Molecules by Terminal Transferase

  Materials
  • 10× BlueJuice gel loading buffer (Invitrogen)
  • 4% to 20% gradient TBE gel, 1.0 mM, 12 well or similar (Invitrogen)
  • 25‐bp DNA Ladder (Invitrogen)
  • 1‐kB DNA Ladder (Invitrogen)
  • Ultrapure 10× TBE buffer (Invitrogen)
  • SYBR Gold Nucleic Acid Gel Stain (Invitrogen)
  • Helicos™ DNA Sample Preparation Reagents Kit (; Helicos BioSciences Corporation, http://www.helicosbio.com, store at –80°C), including:
    • Helicos PolyA Tailing Control Oligonucleotide TR
    • Helicos PolyA Tailing dATP (not all commercial preparations have a functional concentration of dATP as stated; it is important to use a functionally characterized source of dATP like that provided by Helicos BioSciences)
  • Terminal Transferase Kit (New England Biolabs):
    • 2000 U/ml Terminal Transferase Enzyme
    • 2.5 mM CoCl 2
    • 10× Terminal Transferase Buffer
  • 100‐bp DNA Ladder (Invitrogen)
  • 1 mM Biotin‐11‐ddATP (PerkinElmer)
  • 500 mM disodium EDTA ( appendix 22)
  • XCell Surelock Mini‐Cell (Invitrogen)
  • Photodocumentation system compatible with a SYBR Gold photographic filter (AlphaImager EP or HP system; Cell Biosciences)
  • SYBR Gold Nucleic photographic filter (Invitrogen)
  • Nanodrop 1000, 2000, 2000c, or 8000 spectrophotometer (Thermo Fisher Scientific)
  • 0.2‐ml MAXYMum Recovery Thin Wall PCR tubes (Axygen Scientific)
  • 1.5‐ml MAXYMum Recovery tubes (Axygen Scientific)
  • DNA Engine Thermal Cycler (BioRad Laboratories)
  • Aluminum block milled for 0.2‐ml tubes (VWR), prechilled in ice bath

Basic Protocol 3: DNA Sequencing Using the HeliScope™ Genetic Analysis System

  Materials
  • Helicos tSMS™ Reagent Kit (Helicos, cat. no. K‐SMSA)
  • Liquinox (Alconox, cat. no. 1232)
  • Helicos Genetic Analysis System including HeliScope™ Single Molecule Sequencer
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Figures

Videos

Literature Cited

   Axelrod, D., Burghardt, T.P., and Thompson, N.L. 1984. Total internal reflection fluorescence. Annu. Rev. Biophys. Bioeng. 13:247‐268.
   Bowers, J., Mitchell, J., Beer, E., Buzby, P.R., Causey, M., Efcavitch, J.W., Jarosz, M., Krzymanska‐Olejnik, E., Kung, L., Lipson, D., Lowman, G.M., Marappan, S., McInerney, P., Platt, A., Roy, A., Siddiqi, S.M., Steinmann, K., and Thompson, J.F. 2009. Virtual terminator nucleotides for next‐generation DNA sequencing. Nat. Methods 6:593‐595.
   Braslavsky, I., Hebert, B., Kartalov, E., and Quake, S.R. 2003. Sequence information can be obtained from single DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 100:3960‐3964.
   Efcavitch, J.W. and Thompson, J.F. 2010. Single molecule DNA analysis. Ann. Rev. Anal. Chem. 3:109‐128.
   Goren, A., Ozsolak, F., Shoresh, N., Ku, M., Adli, M., Hart, C., Gymrek, M., Zuk, O., Regev, A., Milos, P.M., and Bernstein, B.E. 2010. Chromatin profiling by directly sequencing small quantities of immunoprecipitated DNA. Nat. Methods 7:47‐49.
   Harris, T.D., Buzby, P.R., Babcock, H., Beer, E., Bowers, J., Braslavsky, I., Causey, M., Colonell, J., Dimeo, J., Efcavitch, J.W., Giladi, E., Gill, J., Healy, J., Jarosz, M., Lapen, D., Moulton, K., Quake, S.R., Steinmann, K., Thayer, E., Tyurina, A., Ward, R., Weiss, H., and Xie, Z. 2008. Single‐molecule DNA sequencing of a viral genome. Science 320:106‐109.
   Kahvejian, A., Quackenbush, J., and Thompson, J.F. 2008. What would you do if you could sequence everything? Nat. Biotechnol. 26:1125‐1133.
   Lipson, D., Raz, T., Kieu, A., Jones, D.R., Giladi, E., Thayer, E., Thompson, J.F., Letovsky, S., Milos, P., and Causey, M. 2009. Quantification of the yeast transcriptome by single‐molecule sequencing. Nat. Biotechnol. 27:652‐658.
   Metzker, M.L. 2010. Sequencing technologies: The next generation. Nat. Rev. Genet. 11:31‐46.
   Ozsolak, F., Platt, A.R., Jones, D.R., Reifenberger, J.G., Sass, L.E., McInerney, P., Thompson, J.F., Bowers, J., Jarosz, M., and Milos, P.M. 2009. Direct RNA sequencing. Nature 461:814‐818.
   Ozsolak, F., Goren, A., Gymrek, M., Guttman, M., Regev, A., Bernstein, B.E., and Milos, P.M. 2010. Digital transcriptome profiling from attomole‐level RNA samples. Genome Res. 20:519‐525.
   Pushkarev, D., Neff, N.F., and Quake, S.R. 2009. Single‐molecule sequencing of an individual human genome. Nat. Biotechnol. 27:847‐852.
   Schwartz, J.J. and Quake, S.R. 2007. High density single molecule surface patterning with colloidal epitaxy. Appl. Phys. Lett. 91:083902.
Key References
  Bowers et al., 2009. See above.
  Detailed discussion of the chemistry of the nucleotides.
  Harris et al., 2008. See above.
  The first single‐molecule sequencing of a genome, although accomplished with an older version of the chemistry.
Internet Resources
   http://www.helicosbio.com/Applications/tabid/66/Default.aspx
  Helicos BioSciences Web site for applications and updates
  http://www.covarisinc.com/
  Sites for information on DNA shearing.
  http://www.neb.com/nebecomm/products/productM0348.asp
  http://www.epibio.com/nextera/nextera_tech_overview.asp
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