Preparing DNA Libraries for Multiplexed Paired‐End Deep Sequencing for Illumina GA Sequencers

Mike S. Son1, Ronald K. Taylor1

1 Dartmouth Medical School, Hanover, New Hampshire
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1E.4
DOI:  10.1002/9780471729259.mc01e04s20
Online Posting Date:  February, 2011
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Abstract

Whole‐genome sequencing, also known as deep sequencing, is becoming a more affordable and efficient way to identify SNP mutations, deletions, and insertions in DNA sequences across several different strains. Two major obstacles preventing the widespread use of deep sequencers are the costs involved in services used to prepare DNA libraries for sequencing and the overall accuracy of the sequencing data. This unit describes the preparation of DNA libraries for multiplexed paired‐end sequencing using the Illumina GA series sequencer. Self‐preparation of DNA libraries can help reduce overall expenses, especially if optimization is required for the different samples, and use of the Illumina GA Sequencer can improve the quality of the data. Curr. Protoc. Microbiol. 20:1E.4.1‐1E.4.13. © 2011 by John Wiley & Sons, Inc.

Keywords: multiplexed; paired‐end; deep sequencing; Illumina GA; DNA library

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Chromosomal DNA Purification and Fragmentation
  • Basic Protocol 2: End Repair of Fragmented DNA
  • Basic Protocol 3: 3′ End Modification: Addition of “A” Bases to the 3′ End of the DNA Fragments
  • Basic Protocol 4: Adapter Ligation
  • Basic Protocol 5: Enrichment of Adapter‐Modified DNA Fragments by PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Chromosomal DNA Purification and Fragmentation

  Materials
  • Overnight cultures
  • QIAprep Spin Miniprep kit (Qiagen, cat. no. 27104) containing:
    • EB buffer
  • TE buffer (see recipe)
  • Nebulization buffer (see recipe)
  • Ice and ice bucket
  • 2% low‐melting‐point agarose gel (see recipe)
  • QIAquick PCR Purification kit (Qiagen, cat. no. 28104)
  • NanoDrop spectrophotometer (NanoDrop model no. ND100)
  • 2‐ml microcentrifuge tubes, sterile (USA Scientific, cat. no. 1620‐2700)
  • Nebulizer (Invitrogen, cat. no. K7025‐05)
  • Vacuum tubing (Tygon, cat. no. R3603)
  • Worm drive hose clamp that fits the vacuum tubing
  • Agarose gel electrophoresis setup
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 2: End Repair of Fragmented DNA

  Materials
  • Ice
  • Fragmented DNA sample (see protocol 1)
  • T4 DNA ligase buffer mix (see recipe)
  • Enzyme master mix (see recipe)
  • Sterile double‐distilled water (nuclease‐free)
  • TE buffer (see recipe)
  • Qiagen EB buffer (Qiagen, cat. no. 19086)
  • 200‐µl sterile thin‐walled PCR tubes (Corning, cat. no. 6571)
  • PCR thermal cycler capable of holding 200‐µl PCR tubes with heated lid
  • Petri dishes (Fisher, cat. no. 0875712)
  • Nitrocellulose filter (Millipore, cat. no. VSWP02500)
  • Vacuum dryer centrifuge (Jouan model no. RC10.10)

Basic Protocol 3: 3′ End Modification: Addition of “A” Bases to the 3′ End of the DNA Fragments

  Materials
  • End‐repaired DNA sample ( protocol 2)
  • Klenow buffer (NEB, cat. no. M0212S)
  • 1 mM dATP (NEB, cat. no. M0440S)
  • Klenow fragment (3′ to 5′ exo minus; NEB, cat. no. M0212S)
  • TE buffer (see recipe)
  • Qiagen EB buffer (Qiagen, cat. no. 19086)
  • PCR thermal cycler capable of holding 200‐µl PCR tubes with heated lid
  • Vacuum dryer centrifuge (Jouan model no. RC10.10)

Basic Protocol 4: Adapter Ligation

  Materials
  • 3′ modified DNA sample (see protocol 3)
  • T4 DNA ligase buffer (NEB, cat. no. M0202S)
  • Index PE Adapter oligo mix (Illumina, cat. no. PE400‐1001)
  • T4 DNA ligase (NEB, cat. no. M0202S)
  • 1× TBE (see recipe) for running the gel
  • 6× gel loading dye with gel green (see recipe)
  • 2% low‐melting‐point agarose gel (see recipe)
  • QIAquick Gel Extraction kit (Qiagen, cat. no. 28704)
  • PCR thermal cycler capable of holding 200‐µl PCR tubes with heated lid
  • Transilluminator (Clare Chemical Research Model #DR‐45M)
  • 2.0‐ml sterile microcentrifuge tubes (USA Scientific, cat. no. 1620‐2700)
  • NanoDrop spectrophotometer (NanoDrop model no. ND100)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 5: Enrichment of Adapter‐Modified DNA Fragments by PCR

  Materials
  • Adapter‐ligated DNA sample ( protocol 4)
  • PCR primer InPE1.0 (Illumina, cat. no. PE400‐1001)
  • PCR primer InPE2.0 (Illumina, cat. no. PE400‐1001)
  • PCR index primer (Illumina, cat. no. PE400‐1001)
  • 5× HF Phusion buffer (NEB, cat. no. F‐540S)
  • 10 mM dNTP (NEB, cat. no. N0447S)
  • Phusion DNA polymerase (NEB, cat. no. F‐540S)
  • Sterile double‐distilled water (nuclease‐free)
  • 5% TBE DNA acrylamide gel (BioRad, cat. no. 161‐1109)
  • 6× gel loading dye with gel green (see recipe)
  • 100‐bp marker (NEB, cat. no. N3231S)
  • QIAquick PCR Purification kit (Qiagen, cat. no. 28104)
  • Qiagen EB buffer (Qiagen, cat. no. 19086)
  • 200‐µl sterile thin‐walled PCR tubes (Corning, cat. no. 6571)
  • PCR thermal cycler capable of holding 200‐µl PCR tubes with heated lid
  • DNA acrylamide gel electrophoresis setup
  • NanoDrop spectrophotometer (NanoDrop model no. ND100)
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Figures

Videos

Literature Cited

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