Transposon Insertion Site Sequencing (TIS‐Seq): An Efficient and High‐Throughput Method for Determining Transposon Insertion Site(s) and Their Relative Abundances in a PiggyBac Transposon Mutant Pool by Next‐Generation Sequencing

Yaligara Veeranagouda1, Michel Didier1

1 Molecular Biology and Genomics, Translational Sciences, Sanofi R&D, Chilly‐Mazarin
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.35
DOI:  10.1002/cpmb.47
Online Posting Date:  October, 2017
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Abstract

The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes. Successful screening of PB transposon mutants relies on efficient identification of transposon insertion site(s) (TIS) in mutant cells. However, currently available methods suffer from time‐consuming steps. Here, we present the method for transposon insertion site sequencing (TIS‐Seq) for high‐throughput identification of TIS in transposon mutants. TIS‐Seq provides qualitative and quantitative information on mutants present in a given PB transposon mutant library. TIS‐Seq also facilitates identification of TIS in up to 96 individual/hand‐picked mutants in a single MiniSeq/MiSeq run. TIS‐Seq is a versatile method that can be easily modified to identify TIS from any kind of transposon mutant, as long as one end of the DNA sequence is known. Therefore, TIS‐Seq is a promising method for transposon mutant screening. © 2017 by John Wiley & Sons, Inc.

Keywords: PiggyBac; transposon insertion; transposon mutant; high throughput; mutant screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transposon Insertion Site Sequencing (TIS‐Seq)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transposon Insertion Site Sequencing (TIS‐Seq)

  Materials
  • Genomic DNA (gDNA) from PB transposon mutant libraries (pool of transposon mutants) or individual PB transposon mutants
  • Nuclease‐free water
  • Agilent High‐Sensitivity DNA kit (Agilent Technologies, cat. no. 5067‐4626)
  • 20 U/μl terminal transferase (TdT; New England Biolabs, cat. no. M0315S) with 10× reaction buffer and 10× CoCl 2 (0.25 mM)
  • dCTP/ddCTP mix: 9.5 mM dCTP + 0.5 mM ddCTP (each from Sigma‐Aldrich)
  • MagJET NGS Cleanup and Size Selection Kit (Thermo Fisher Scientific, cat. no. K2821)
  • NEBNext High‐Fidelity 2× PCR Master Mix (New England Biolabs, cat. no. M0541S)
  • Primers (see Tables 21.35.1 and 21.35.2)
  • DNA Clean & Concentrator‐25 Kit with capped columns (Zymo Research, cat. no. D4033) or equivalent
  • Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, cat. no. Q32851)
  • microTUBEs (AFA Fiber Pre‐Slit Snap‐Cap, 6 × 16 mm; Covaris, cat. no. 520045)
  • M220 Focused‐ultrasonicator (Covaris) with SonoLab software
  • 1.5‐ml nuclease‐free microcentrifuge tubes
  • Vacuum concentrator
  • NanoDrop 2000 (or UV‐vis) spectrophotometer
  • Agilent 2100 Bioanalyzer system (Agilent Technologies)
  • Agilent 2100 chip priming station and IKA vortex mixer (Agilent Technologies)
  • Vortex mixer/multi‐vial vortex shaker
  • Thermocycler
  • MagJET Separation Rack (Thermo Fisher Scientific, cat. no. MR02)
  • Qubit 2.0 (or above) Fluorometer (Thermo Fisher Scientific)
  • Illumina sequencer (MiniSeq or NextSeq500) and appropriate sequencing kits with 75 or 150 cycles
  • Additional reagents and equipment for agarose gel electrophoresis (optional)
NOTE: Use DNase/RNase‐free water in all recipes and protocol steps.
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Figures

Videos

Literature Cited

Literature Cited
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