Microbiota Analysis Using an Illumina MiSeq Platform to Sequence 16S rRNA Genes

Alexis Rapin1, Céline Pattaroni2, Benjamin J. Marsland2, Nicola L. Harris1

1 Global Health Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, 2 Faculty of Biology and Medicine, University of Lausanne, Lausanne
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.29
Online Posting Date:  June, 2017
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Abstract

The microbiota have been shown to play an important role in diverse biological processes including immunity, metabolism, and digestion. Assessing the exact composition of the microbiota has proven challenging due to the often unknown growth specificities of its members, and culture‐based approaches typically fail to capture the complete diversity of microorganisms present. Next Generation Sequencing (NGS) methods provide an efficient means to gather information about cultured and uncultured members of the microbiota. This article provides a method to characterize bacterial communities in terms of species composition using high‐throughput sequencing. Briefly, by extracting the entire DNA content of a microbiota sample and performing a targeted high‐throughput sequencing of the 16S rRNA gene, a phylogenetic marker for prokaryotes, prediction of the composition of the entire bacterial community is made possible. © 2017 by John Wiley & Sons, Inc.

Keywords: MiSeq; microbiota; next generation sequencing; 16S rRNA

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Bacterial 16S rRNA Sequencing for Bacterial Communities Present in Intestinal Contents of Mice, or From Fecal Samples Collected From Mice or Humans
  • Alternate Protocol 1: Bacterial 16S rRNA Sequencing of Bacterial Communities Present on the Surface of Mouse or Human Skin
  • Support Protocol 1: 16S rRNA PCR Quality Check
  • Support Protocol 2: Purification, Quantification, and Pooling of 16S rRNA Amplicon Libraries
  • Support Protocol 3: Purification of 16S rRNA Amplicon Libraries Using Gel Electrophoresis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Bacterial 16S rRNA Sequencing for Bacterial Communities Present in Intestinal Contents of Mice, or From Fecal Samples Collected From Mice or Humans

  Materials
  • Stool donor: human or mouse
  • Stool collection kit for human specimens
  • PowerSoil DNA Isolation Kit (MO BIO, cat. no. 12888‐50/100)
  • FastPrep‐24 beater (MP Biomedicals) or similar
  • Nuclease‐free H 2O
  • Template DNA
  • 10 μM 16S rRNA universal primers (see Table 17.0.4000 and Figure ):
    • 10 μM reverse PCR primer (see Figure and Table 17.0.4000 for details; can be synthesized on demand by a commercial oligonucleotide supplier)
    • 10 μM forward PCR primer (see Figure and Table 17.0.4000 for details; can be synthesized on demand by a commercial oligonucleotide supplier)
  • 2× NEBNext High‐Fidelity 2× Q5 PCR Master Mix (from High‐Fidelity Polymerase Kit; New England Biolabs, cat. no. E0555)
  • DNA 5K Kit (PerkinElmer, cat. no. CLS760675)
  • DNA‐binding magnetic beads (Agencourt AMPure XP beads, Beckman Coulter, cat. no. A63880)
  • 80% (v/v) ethanol
  • 10 mM Tris·Cl, pH 7.5
  • Qubit High Sensitivity kit (Thermo Fisher Scientific, cat. no. Q32851) or Quant‐iT PicoGreen dsDNA reagent (Thermo Fisher Scientific, cat. no. P11495/P7581)
  • TE buffer, pH 8 (Tris·Cl, pH 8/1 mM disodium EDTA in ultrapure H 2O)
  • dsDNA standards at 0 ng/μl and 10 ng/μl (included in Qubit High Sensitivity kit)
  • MiSeq Reagent Kit v2 (500‐cycles; Illumina, cat. no. MS‐102‐2003)
  • 100 μM sequencing primers (see Table 17.0.4000 and Figure )
  • 1 N NaOH
  • PhiX Control v3 (Illumina, cat. no. 15017666)
  • 1.5‐ and 2‐ml capped polypropylene microcentrifuge tubes
  • 50‐ml syringe
  • Gavage needle
  • Tweezers
  • Scissors
  • Pins
  • Microscopy cover slip
  • Sterile scalpel blades or sterile toothpicks
  • PCR plate or PCR tubes with seals, adapted for thermal cycler
  • Thermal cycler
  • LabChip GX capillary electrophoresis machine (PerkinElmer)
  • Magnetic rack for 1.5‐ml tubes
  • Qubit fluorometer (Thermo Fisher Scientific, cat. no. Q33216)
  • Thin‐walled 0.5‐μl Qubit assay tubes (Thermo Fisher Scientific, cat. no. Q32856)
  • MiSeq sequencer (Illumina)
  • 1.5‐ml syringes with ∼80‐mm‐long needles or Pasteur pipets
  • Additional reagents and equipment for PCR (Kramer & Coen, ), PCR quality check ( protocol 3), and preparing multiplexed 16S libraries pool ( protocol 4)

Alternate Protocol 1: Bacterial 16S rRNA Sequencing of Bacterial Communities Present on the Surface of Mouse or Human Skin

  Materials
  • ESwab Liquid Amies Collection System, nylon swab (Copan, cat. no. 493C02)
  • Lysozyme solution (see recipe)
  • Proteinase K solution (see recipe)
  • 100% ethanol
  • QIAmp DNA Mini Kit (Qiagen, cat. no. 51306)
  • 1.5‐ and 2‐ml capped polypropylene microcentrifuge tubes
  • Animal trimmer
  • Orbital shaker and heating block suitable for 2‐ml tubes
  • PCR plate or PCR tubes with seals adapted for thermal cycler
  • Additional reagents and equipment for mouse anesthesia (Adams & Pacharinsak, )

Support Protocol 1: 16S rRNA PCR Quality Check

  Materials
  • Gel loading dye (NEB, cat. no. B7021)
  • 2‐log DNA ladder (NEB, N3200)
  • 2% TAE agarose gel (prepared using an established protocol; Voytas, ) containing DNA‐binding dye (GelRed Nucleic Acid Gel Stain, Biotium, cat. no. 41003)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Support Protocol 2: Purification, Quantification, and Pooling of 16S rRNA Amplicon Libraries

  Materials
  • PCR products from step 4 of the protocol 1Basic Protocol
  • DNA‐binding magnetic beads (Agencourt AMPure XP beads, Beckman Coulter, cat. no. A63880)
  • 80% (v/v) ethanol
  • 10 mM Tris·Cl, pH 7.5
  • Quant‐iT PicoGreen dsDNA reagent (Thermo Fisher Scientific, cat. no. P11495/P7581)
  • dsDNA standards
  • TE buffer, pH 8 (Tris·Cl, pH 8/1 mM disodium EDTA in ultapure H 2O)
  • Magnetic rack for 96‐well PCR plate
  • Flat‐bottom, transparent 96‐well plate
  • Plate reader with 480 nm excitation and 530 nm emission reading capability

Support Protocol 3: Purification of 16S rRNA Amplicon Libraries Using Gel Electrophoresis

  Materials
  • PCR products from step 4 of the protocol 1Basic Protocol
  • Gel loading dye (NEB, cat. no. B7021)
  • 2% TAE agarose gel with 50 μl well (prepared using an established protocol; Voytas, ) and containing DNA‐binding dye (GelRed Nucleic Acid Gel Stain; Biotium, cat. no. 41003)
  • Gel clean‐up system (Wizard SV Gel and PCR Clean‐Up System; Promega, cat. no. A9281)
  • Blue light or UV table for gel visualization and cutting
  • Scalpel
  • 2‐ml capped polypropylene microcentrifuge tubes
  • Heating block suitable for 2‐ml tubes (for gel clean‐up system)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )
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Figures

Videos

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Internet Resources
  http://support.illumina.com/sequencing/sequencing_instruments/miseq/documentation.html
  Illumina MiSeq documentation, including preparation guides and kit documentation.
  https://support.illumina.com/bulletins/2016/06/how‐to‐create‐fastq‐files‐for‐index‐reads‐in‐miseq‐reporter.html
  Illumina support bulletin for setting up the MiSeq Reporter Software in order to produce FASTQ files for Index reads.
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