Streptococcus pneumoniae Supragenome Hybridization Arrays for Profiling of Genetic Content and Gene Expression

Anagha Kadam1, Benjamin Janto2, Rory Eutsey3, Joshua P. Earl2, Evan Powell3, Margaret E. Dahlgren3, Fen Z. Hu2, Garth D. Ehrlich2, N. Luisa Hiller3

1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, 2 Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, 3 Center of Excellence in Biofilm Research, Allegheny Health Network, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9D.4
DOI:  10.1002/9780471729259.mc09d04s36
Online Posting Date:  February, 2015
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Abstract

There is extensive genomic diversity among Streptococcus pneumoniae isolates. Approximately half of the comprehensive set of genes in the species (the supragenome or pangenome) is present in all the isolates (core set), and the remaining is unevenly distributed among strains (distributed set). The Streptococcus pneumoniae Supragenome Hybridization (SpSGH) array provides coverage for an extensive set of genes and polymorphisms encountered within this species, capturing this genomic diversity. Further, the capture is quantitative. In this manner, the SpSGH array allows for both genomic and transcriptomic analyses of diverse S. pneumoniae isolates on a single platform. In this unit, we present the SpSGH array, and describe in detail its design and implementation for both genomic and transcriptomic analyses. The methodology can be applied to construction and modification of SpSGH array platforms, as well to other bacterial species as long as multiple whole‐genome sequences are available that collectively capture the vast majority of the species supragenome. © 2015 by John Wiley & Sons, Inc.

Keywords: supragenome; pangenome; gene array; transcriptional profiling; gene expression; genome content; Streptococcus pneumoniae

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Probe Design for SpSGH Array
  • Basic Protocol 2: SpSGH Array to Determine Gene Content
  • Basic Protocol 3: Data Analysis of SpSGH Array to Determine Gene Content
  • Basic Protocol 4: SpSGH Array for Gene‐Expression Profiling
  • Basic Protocol 5: Data Analysis of SpSGH Array for Gene‐Expression Profiling
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Probe Design for SpSGH Array

  Materials
  • WGS of multiple strains that capture the diversity of the set of interest
  • Rapid Annotations with Subsystems Technology (RAST; http://rast.nmpdr.org/)
  • FASTA36 from the FASTA package (http://faculty.virginia.edu/wrpearson/fasta/fasta36/)
  • Scripts for gene clustering (available from the authors on request, or at https://github.com/jpearl01/)
  • Array manufacturer's probe‐design tool

Basic Protocol 2: SpSGH Array to Determine Gene Content

  Materials
  • Bacterial strain of interest
  • Standard media for bacterial growth: Columbia broth (unit 13.1) is used for S. pneumoniae and BHI broth (e.g., BD Difco) is used for H. influenzae
  • Pneumococcal cell lysis cocktail: TE buffer ( appendix 2A) containing lysozyme (15 ml/ml), mutanolysin (30 µg/ml; Sigma, cat. no. M9901), and proteinase K (20 mg/ml)
  • 1% agarose gel (Voytas, ) prepared in TAE buffer
  • 4 mg/ml RNaseA
  • 20 mg/ml proteinase K
  • gDNA Cy‐3 labeling reagents from the array manufacturer (e.g., NimbleGen, Agilent)
  • NimbleGen One Color DNA Labeling Kit (Gene Expression Arrays version 6.0)
  • Hybridization and washing reagents for SGH array (e.g., NimbleGen, Agilent)
  • Centrifuge that allows harvesting cells from a 15‐ml culture volume
  • NanoDrop 1000 UV microspectrophotometer (http://www.nanodrop.com)
  • Vacuum concentrator (e.g., SpeedVac evaporator)
  • Uv/vis spectrophotometer
  • Heat block
  • Proprietary mixing device that is designed to align and adhere to the surface of the array (NimbleGen; referred to as “mixer” in catalog)
  • SGH slide containing arrays (NimbleGen)
  • Hybridization station (NimbleGen)
  • Fluorescent scanner (e.g., Molecular Devices Axon GenePix 4200AL)
  • NimbleScan (NimbleGen) or equivalent imaging software
  • Additional reagents and equipment for preparation of genomic DNA from bacteria (Wilson, ) and agarose gel electrophoresis (Voytas, )

Basic Protocol 3: Data Analysis of SpSGH Array to Determine Gene Content

  Materials
  • Scans from arrays on the slide ( protocol 2)
  • Slide scanning software provided by the array manufacturer (e.g., NimbleScan)

Basic Protocol 4: SpSGH Array for Gene‐Expression Profiling

  Materials
  • Bacterial strain of interest
  • Standard medium for bacterial growth: Columbia broth (unit 13.1) is used for S. pneumoniae
  • RNAProtect Bacterial Reagent (Qiagen)
  • RNeasy Mini Kit (Qiagen) for RNA extraction
  • Pneumococcal cell lysis cocktail: TE buffer ( appendix 2A) containing lysozyme (15 ml/ml), mutanolysin (30 µg/ml; Sigma, cat. no. M9901), and proteinase K (20 mg/ml)
  • Lysis buffer RLTplus (Qiagen)
  • 2 U/µl Turbo DNase (Ambion)
  • SuperScript III First‐Strand Synthesis SuperMix kit (Invitrogen)
  • SuperScript Double‐Stranded cDNA Synthesis kit (Invitrogen)
  • 4 mg/ml RNaseA
  • 25:24:1 phenol:chloroform:isoamyl alcohol ( appendix 2A)
  • Centrifuge that allows harvesting cells from a 15‐ml culture volume
  • gDNA eliminator column (Qiagen)Agilent Bioanalyzer and RNA 6000 Nano Kit
  • NanoDrop1000 microspectrophotometer
  • Additional reagents and equipment for the polymerase chain reaction (PCR; Kramer and Coen, ), precipitation of DNA (Moore and Dowhan, ), and Cy3 DNA labeling, hybridization, washing, and scanning ( protocol 2)

Basic Protocol 5: Data Analysis of SpSGH Array for Gene‐Expression Profiling

  Materials
  • Slide scanning software provided by the array manufacturer (such as NimbleScan)
  • NCBI‐BLAST
  • Software of choice for transcriptomic analysis (e.g., CyberT, http://cybert.microarray.ics.uci.edu/; SAM, http://statweb.stanford.edu/∼tibs/SAM/faq.html; TM4/MeV, http://www.tm4.org/mev.html)
NOTE: If the probe set of choice in selected by hybridization, also refer to protocol 2
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Figures

Videos

Literature Cited

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