Methods for Generating Shotgun and Mixed Shotgun/Paired‐End Libraries for the 454 DNA Sequencer

Graham Wiley1, Simone Macmil1, Chunmei Qu1, Ping Wang1, Yanbo Xing1, Doug White1, Jianfeng Li1, James D. White1, Alexander Domingo2, Bruce A. Roe1

1 University of Oklahoma, Norman, Oklahoma, 2 Roche Diagnostics, Indianapolis, Indiana
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 18.1
DOI:  10.1002/0471142905.hg1801s61
Online Posting Date:  April, 2009
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Abstract

With the introduction of massively parallel, microminiature‐based instrumentation for DNA sequencing, robust, reproducible, optimized methods are needed to prepare the target DNA for analysis using these high‐throughput approaches because the cost per instrument run is orders of magnitude more than for typical Sanger dideoxynucleotide sequencing on fluorescence‐based capillary systems. The methods provided by the manufacturer for genome sequencing using the 454/Roche GS‐20 and GS‐FLX instruments are robust. However, in an effort to streamline them for automation, we have incorporated several novel changes and deleted several extraneous steps. As a result of modifying these sample preparation protocols, the number of manual manipulations has also been minimized, and the overall yields have been improved for both shotgun and mixed shotgun/paired‐end libraries. Curr. Protoc. Hum. Genet. 61:18.1.1‐18.1.21. © 2009 by John Wiley & Sons, Inc.

Keywords: pyrosequencing; shotgun; paired‐end; DNA Library

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Shotgun Library Preparation
  • Basic Protocol 2: Shotgun/Paired‐End Library Preparation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Shotgun Library Preparation

  Materials
  • Isopropanol, chilled to −80°C
  • GS DNA Library Preparation Kit (Roche), including:
    • Nebulizer, with condenser and aspiration tubes
    • TE buffer
    • Nebulization buffer
    • 10× polishing buffer
    • BSA
    • ATP
    • dNTP mix
    • T4 polynucleotide kinase
    • T4 DNA polymerase
    • 2× ligase buffer
    • A and B adaptors
    • Ligase
    • Molecular biology grade water
    • 10× Fill‐In polymerase buffer
    • Fill‐In polymerase
  • 3 to 5 µg purified, double‐stranded DNA: e.g., see Roe et al. ( ), Roe ( ), appendix 3B, appendix 3C, or use a commercially available DNA isolation kit
  • Compressed nitrogen (research grade)
  • MinElute PCR Purification Kit (Qiagen), including:
    • PBI buffer
    • MinElute centrifuge columns
    • PE buffer
    • EB buffer
  • AMPure SPRI beads (Agencourt)
  • 70% (v/v) ethanol
  • Dual‐stage regulator on the nitrogen gas cylinder connected to the nebulizer using:
    • ¼‐in. (6.35‐mm) hose barb fitting with ¼‐in. (6.35‐mm) NPT female compression fitting
    • ¼‐in. (6.35‐mm) i.d. flexible tubing, with stainless steel screw clamp
  • 1.5‐ml microcentrifuge tubes
  • Microtiter plate for automated microfluidics system (Caliper)
  • Automated microfluidics system: AMS‐90 and SE30 DNA LabChip (Caliper)
  • Magnetic particle concentrator (Invitrogen)
  • Vacuum drying system (e.g., model 5831; National Appliance)
NOTE: Most of the reagents in this protocol were obtained as components of the GS DNA Library Preparation Kit (Roche). The components of the kit are based on those described in the supplement to Margulies et al. ( ).NOTE: All microcentrifugations are performed at room temperature.

Basic Protocol 2: Shotgun/Paired‐End Library Preparation

  Materials
  • 3‐5 µg purified, double‐stranded DNA in TE buffer: e.g., see Roe et al. ( ), Roe ( ), appendix 3B, appendix 3C, or use a commercially available DNA isolation kit
  • TE buffer ( appendix 2D)
  • AMPure SPRI beads (Agencourt)
  • 70% (v/v) ethanol
  • MinElute PCR purification kit (Qiagen), including:
    • EB buffer
    • MinElute centrifuge columns
    • PBI buffer
    • PE buffer
  • 32 mM S‐adenosylmethionine (SAM) stock (New England Biolabs)
  • Molecular biology grade water
  • 10× EcoRI methylase buffer and 40,000 U/ml EcoRI methylase (New England Biolabs)
  • 10 mg/ml bovine serum albumin (BSA; New England Biolabs)
  • 30,000 U/ml T4 polynucleotide kinase and 10× polynucleotide kinase buffer (USB)
  • 10 mM (each) dNTP mix (see appendix 2D)
  • 3000 U/ml T4 DNA polymerase (New England Biolabs)
  • 2× Rapid Ligase buffer and Rapid Ligase (Roche)
  • Hairpin linker (GS DNA Paired End Adaptor Kit, Roche)
  • 100 mM ATP ( appendix 2D)
  • 5000 U/ml lambda exonuclease (New England Biolabs)
  • 10,000 U/ml T7 exonuclease (New England Biolabs)
  • 5000 U/ml exonuclease I (New England Biolabs)
  • 20,000 U/ml EcoRI
  • 10× NEBuffer 4 (New England Biolabs)
  • Isopropanol, chilled to −80°C
  • GS DNA Library Preparation Kit (Roche), including:
    • Nebulizer, with condenser and aspiration tubes
    • TE buffer
    • Nebulization buffer
    • Polishing buffer
    • BSA
    • ATP
    • dNTP mix
    • T4 polynucleotide kinase
    • T4 DNA polymerase
    • 2× ligase buffer
    • A and B adaptors
    • Ligase
    • Molecular biology grade water
    • 10× Fill‐In polymerase buffer
    • Fill‐In polymerase
  • Compressed nitrogen (research grade)
  • 1.5‐ml microcentrifuge tube
  • GeneMachines HydroShear apparatus, equipped with the~HydroShear large assembly and HydroShear syringe (Genomic Solutions)
  • Magnetic particle concentrator (Invitrogen)
  • Vacuum drying system (e.g., model 5831; National Appliance)
  • Microtiter plate for the automated microfluidics system (Caliper)
  • Automated microfluidics system: AMS‐90 and SE30 DNA LabChip (Caliper)
  • Thermal cycler or heating block
  • 12°C refrigerator
  • Dual‐stage regulator on the nitrogen gas cylinder connected to the nebulizer using:
    • ¼‐in. (6.35‐mm) hose barb fitting with ¼‐in. (6.35‐mm) NPT female compression fitting
    • ¼‐in. (6.35‐mm) i.d. flexible tubing, with stainless steel screw clamp
NOTE: Most of the reagents used in this protocol were obtained as components of the GS DNA Library Preparation Kit (Roche). The components of the kit are based on those described in the supplement to Margulies et al. ( ).NOTE: All microcentrifugations are performed at room temperature.
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Figures

Videos

Literature Cited

Literature Cited
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   Oefner, P.J., Hunicke‐Smith, S.P., Chiang, L., Dietrich, F., Mulligan, J., and Davis, R.W. 1996. Efficient random subcloning of DNA sheared in a recirculating point‐sink flow system. Nucleic Acids Res. 24:3879‐3886.
   Roe, B. 2004. Shotgun library construction for DNA sequencing In Methods in Molecular Biology, vol 255: Bacterial Artificial Chromosomes, Volume 1: Library Construction, Physical Mapping, and Sequencing (S. Zhao and M. Stodolsky, eds.) pp. 171‐187. Humana Press, Totowa, New Jersey.
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   Ronaghi, M., Uhlén, M. and Nyrén, P. 1998. DNA Sequencing:A sequencing method based on real‐time pyrophosphate. Science 281:363‐365.
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