Genome‐Wide Fitness and Genetic Interactions Determined by Tn‐seq, a High‐Throughput Massively Parallel Sequencing Method for Microorganisms

Tim van Opijnen1, David W. Lazinski2, Andrew Camilli2

1 Department of Biology, Boston College, Chestnut Hill, Massachusetts, 2 Department of Molecular Biology and Microbiology, School of Medicine, Tufts University, and Howard Hughes Medical Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.16
DOI:  10.1002/0471142727.mb0716s106
Online Posting Date:  April, 2014
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Abstract

The lagging annotation of bacterial genomes and the inherent genetic complexity of many phenotypes is hindering the discovery of new drug targets and the development of new antimicrobial agents and vaccines. This unit presents Tn‐seq, a method that has made it possible to quantitatively determine fitness for most genes in a microorganism and to screen for quantitative genetic interactions on a genome‐wide scale and in a high‐throughput fashion. Tn‐seq can thus direct studies on the annotation of genes and untangle complex phenotypes. The method is based on the construction of a saturated transposon insertion library. After library selection, changes in the frequency of each insertion mutant are determined by sequencing flanking regions en masse. These changes are used to calculate each mutant's fitness. The method was originally developed for the Gram‐positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia and meningitis, but has now been applied to several different microbial species. Curr. Protoc. Mol. Biol. 106:7.16.1‐7.16.24 . © 2014 by John Wiley & Sons, Inc.

Keywords: transposon sequencing; Tn‐seq; Streptococcus pneumoniae ; Vibrio cholerae ; genome‐wide fitness; genetic interactions; transposon mutagenesis; massively parallel sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation of a magellan6 Transposon Library in S. pneumoniae by In Vitro Transposition
  • Support Protocol 1: Purification of Mariner Transposase
  • Basic Protocol 2: Transformation of S. pneumoniae with magellan6 Transposon Insertion Library
  • Basic Protocol 3: Transposon Library Selection and Sample Preparation for Illumina Sequencing
  • Alternate Protocol 1: Transposon Library Construction in V. cholerae by Conjugation
  • Alternate Protocol 2: General Method for Tn‐seq Sample Preparation for Illumina Sequencing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation of a magellan6 Transposon Library in S. pneumoniae by In Vitro Transposition

  Materials
  • S. pneumoniae genomic DNA (obtained using a Qiagen Blood & Tissue Kit)
  • pMagellan6 DNA (available on request; andrew.camilli@tufts.edu)
  • 2× Buffer A (see recipe)
  • MarC9 mariner transposase (see Support Protocol)
  • 10× Buffer B (see recipe)
  • 10× (10 mg/ml) bovine serum albumin (BSA; New England Biolabs)
  • 2 mM dNTPs ( )
  • 3 U/µl T4 DNA polymerase (New England Biolabs)
  • 2.6 mM nicotine adenine dinucleotide (NAD)
  • 10 U/µl E. coli DNA ligase (New England Biolabs)
  • 30° and 75°C heating blocks
  • 12° and 16°C cooling blocks
  • Additional reagents and equipment for ethanol extraction (unit ) and DNA quantitation ( )

Support Protocol 1: Purification of Mariner Transposase

  Materials
  • E. coli pMalC9 strain (Lampe et al., ; available on request from )
  • LB/ampicillin: LB broth (unit ) supplemented with 100 µg/ml ampicillin
  • Isopropyl‐β‐D‐thiogalactopyranoside (IPTG)
  • Column buffer (see recipe)
  • Amylose resin (New England Biolabs)
  • Transposase wash buffer (TWB; see recipe)
  • Transposase elution buffer (TEB; see recipe)
  • 37°C, 5% CO 2 incubator
  • 15‐ml centrifuge tube
  • 1.5‐ml microcentrifuge tubes

Basic Protocol 2: Transformation of S. pneumoniae with magellan6 Transposon Insertion Library

  Materials
  • S. pneumoniae strain (e.g., D39 or TIGR4)
  • Sheep's blood agar plates (see recipe), with and without 200 µg/ml spectinomycin
  • Todd‐Hewitt yeast (THY): 30 g/liter Todd‐Hewitt base (Becton Dickinson, cat. no. 249240), pH 7.3, with 15% yeast extract
  • Oxyrase (Oxyrase, cat. no. OB‐0010)
  • THY containing 12% (v/v) glycerol
  • 1 N HCl
  • 20% (w/v) glycine in distilled water
  • 1 N NaOH
  • 8% (w/v) bovine serum albumin (BSA)
  • 1 M CaCl 2
  • 350 ng/µl competence stimulating peptide (CSP; AnaSpec) appropriate to the S. pneumoniae strain used (e.g., CSP1 for D39, CSP2 for TIGR4)
  • Transposon library DNA (see protocol 1)
  • THY containing 20 µg/ml spectinomycin
  • 37°C, 5% CO 2 incubator
  • 1.5‐ml microcentrifuge tubes
  • Large (16‐mm‐diameter) and small (13‐mm‐diameter) glass culture tubes
  • Spectrophotometer
  • 37°C heating block

Basic Protocol 3: Transposon Library Selection and Sample Preparation for Illumina Sequencing

  Materials
  • Transposon library starter culture (see protocol 3)
  • Todd‐Hewitt yeast (THY): 30 g/liter TH base (Becton Dickinson, cat. no. 249240), pH 7.3, with 15% yeast extract
  • Spectinomycin (Sigma‐Aldrich)
  • Oxyrase (Oxyrase, cat. no. OB‐0010)
  • DNeasy Blood & Tissue Kit (Qiagen)
  • Adapter oligonucleotides (Table 7.16.1)
  • 1 mM Tris·Cl, pH 8.3 ( )
  • 2 U/µl MmeI restriction enzyme (New England Biolabs, cat. no. R0637S)
  • 32 mM S‐adenosyl methionine (SAM; New England Biolabs)
  • 10× Buffer 4 (New England Biolabs)
  • Calf intestinal phosphatase (CIP; New England Biolabs, cat. no. M0290S)
  • 400 U/µl T4 DNA ligase and buffer (New England Biolabs)
  • PFU Ultra polymerase and buffer (Stratagene)
  • 25 mM dNTP mix
  • 10 µM each PCR primer (Table 7.16.2): primer P1_M6_MmeI and Gex PCR Primer 2
  • GelGreen DNA stain (Biotium)
  • 50‐bp DNA ladder
  • QIAquick Gel Extraction Kit (Qiagen cat. no. 28704)
  • Large (16‐mm‐diameter) glass culture tubes
  • Spectrophotometer
  • 37°C, 5% CO 2 incubator
  • 37° and 96°C heating blocks
  • 16°C cooling block
  • Thermal cycler
  • UV transilluminator
  • Bioanalyzer (Agilent)
  • Additional reagents and equipment for isolation of genomic DNA from bacteria (unit ), phenol/chloroform extraction and ethanol precipitation of DNA (unit ), microvolume DNA quantitation ( ), and agarose gel electrophoresis (unit )

Alternate Protocol 1: Transposon Library Construction in V. cholerae by Conjugation

  Materials
  • Streptomycin‐resistant V. cholerae recipient strain
  • E. coli donor strain Sm10λpir carrying pUTmTn5Km2 (Merrell et al., ; available on request from )
  • LB broth (unit ), unsupplemented and supplemented individually with 130 µg/ml kanamycin or 100 µg/ml streptomycin
  • LB agar plates (unit ), unsupplemented and supplemented with 100 µg/ml streptomycin plus 130 µg/ml kanamycin
  • 80% (w/v) glycerol
  • 37°C incubator
  • Glass plating rod
  • 1.5‐ml or larger microcentrifuge tubes or cryovials
  • Additional reagents and equipment for replica plating (unit )

Alternate Protocol 2: General Method for Tn‐seq Sample Preparation for Illumina Sequencing

  Materials
  • PCR and sequencing primers for mTn5 insertion libraries (Table 7.16.3)
  • 1 mM Tris·Cl, pH 8.3 ( )
  • V. cholerae transposon library (see protocol 5)
  • DNeasy Blood & Tissue Kit (Qiagen)
  • GelGreen DNA stain (Biotium)
  • QIAquick PCR Purification kit (Qiagen)
  • dCTP
  • ddCTP
  • Terminal deoxynucleotidyl transferase (TdT) and 5× reaction buffer (Promega)
  • DTR Gel Filtration Cartridge (Performa)
  • 25 mM dNTP mix
  • Easy‐A Cloning Enzyme with 10× reaction buffer (Agilent)
  • 1.5‐ml microcentrifuge tubes
  • Branson high‐intensity cuphorn sonifier (Branson)
  • 37° and 75°C heating blocks
  • Thermal cycler
  • Additional reagents and equipment for library selection and analysis of fitness (see protocol 4), microvolume DNA quantitation ( ), and agarose gel electrophoresis (unit )
Table 7.6.3   MaterialsPCR and Illumina Sequencing Primers for Processing mTn5 Insertion Libraries

Primer Sequence (5′ to 3′) Purification
olj491 a ACC TGC AGG CAT GCA AGC TTC GGC C Standard desalting
olj376 b, c GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TGG GGG GGG GGG GGG GG Standard desalting
olj492 a, c AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCG CGG CCG CAC TTG TGT ATA AGA GT HPLC
Barcode primer c, d CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T HPLC
Custom sequencing primer HK89 e ACA CTC TTT CGC GGC CGC ACT TGT GTA TAA GAG TCA G Standard desalting

 aNested primers specific to one end of mTn5; the underlined sequence is complementary to mTn5.
 bThe underlined sequence is complementary to the poly‐dC tail.
 cThe italicized regions are Illumina‐specific sequences necessary for annealing to oligos present in the flow cell.
 dNNNNNN represents the reverse complement of the barcode and is varied with each sample.
 eSpecific to the mTn5 inverted repeat end sequence.
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Figures

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Literature Cited

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