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 Microbiology
Unit Number:  Unit 1E.3
DOI:  10.1002/9780471729259.mc01e03s36
Online Posting Date:  February, 2015
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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. © 2015 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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Basic Protocol 1: Generation of a magellan6 Transposon Library in S. pneumoniae by In Vitro Transposition

  • S. pneumoniae genomic DNA (obtained using a Qiagen Blood & Tissue Kit)
  • pMagellan6 DNA (available on request; )
  • 2× Buffer A (see recipe)
  • MarC9 mariner transposase (see protocol 2Support 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 (Moore and Dowhan, ) and DNA quantitation (Gallagher, )

Support Protocol 1: Purification of Mariner Transposase

  • E. coli pMalC9 strain (Lampe et al., ; available on request from )
  • LB/ampicillin: LB broth (appendix 4a; Stevenson, ) 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

  • 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

  • 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 1.3.1)
  • 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 1.3.2): 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 (DNeasy Blood & Tissue Kit; also see Wilson, 1997), phenol/chloroform extraction and ethanol precipitation of DNA (Moore and Dowhan, ), microvolume DNA quantitation (Desjardins and Conklin, ), and agarose gel electrophoresis (Voytas, )

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

  • Streptomycin‐resistant V. cholerae recipient strain
  • E. coli donor strain Sm10λpir carrying pUTmTn5Km2 (Merrell et al., ; available on request from )
  • LB broth ( ; Stevenson, ), unsupplemented and supplemented individually with 130 μg/ml kanamycin or 100 μg/ml streptomycin
  • LB agar plates ( ; Stevenson, ), 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 (Elbing and Brent, )

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

  • PCR and sequencing primers for mTn5 insertion libraries (Table 1.3.3)
  • 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 (Desjardins and Conklin, ), and agarose gel electrophoresis (Voytas, )
Table 1.0.3   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
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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