A Microbiome DNA Enrichment Method for Next‐Generation Sequencing Sample Preparation

Erbay Yigit1, George R. Feehery1, Bradley W. Langhorst1, Fiona J. Stewart1, Eileen T. Dimalanta1, Sriharsa Pradhan1, Barton Slatko1, Andrew F. Gardner1, James McFarland1, Christine Sumner1, Theodore B. Davis1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.26
DOI:  10.1002/cpmb.12
Online Posting Date:  July, 2016
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“Microbiome” is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before sequencing results in greater sequence depth of the intended microbiome target population. This unit describes a novel method of microbial DNA enrichment in which methylated host DNA such as human genomic DNA is selectively bound and separated from microbial DNA before next‐generation sequencing (NGS) library construction. This microbiome enrichment technique yields a higher fraction of microbial sequencing reads and improved read quality resulting in a reduced cost of downstream data generation and analysis. © 2016 by John Wiley & Sons, Inc.

Keywords: DNA sequencing; microbiome; metagenomics DNA enrichment; methylation; next‐generation sequencing; NGG; target enrichment

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Table of Contents

  • Introduction
  • Basic Protocol 1: Microbiome DNA Enrichment for Next‐Generation Sequencing Sample Preparation
  • Alternate Protocol 1: Eluting Captured Host DNA
  • Alternate Protocol 2: Ethanol Precipitation of DNA
  • Alternate Protocol 3: Quantitation and Validation of Enrichment by Agarose Gel Electrophoresis
  • Commentary
  • Figures
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Basic Protocol 1: Microbiome DNA Enrichment for Next‐Generation Sequencing Sample Preparation

  • Sample: DNA samples should be in 1× TE buffer, be free of proteins, proteinase, SDS and organic solvents, be at least 15 kb or greater in size and free of small molecular weight fragments
  • DNA marker (Lambda DNA‐HindIII Digest, NEB, cat. no. N3012)
  • 6× Gel Loading Dye Blue (NEB, cat. no. B7021)
  • NEBNext kit (NEB, cat. no. E2612) containing:
    • NEBNext protein A magnetic beads
    • NEBNext MBD2‐Fc protein
    • NEBNext Bind/wash buffer (5×)
    • 16S rRNA universal gene bacterial control primers (20 μM each)
    • 5‐GCTTGACGGGCGGTGT (reverse)
  • RPL30 human DNA control primers (20 μM each)
  • Ice
  • Nuclease‐free water
  • Agencourt AMPure XP beads
  • 80% ethanol (freshly prepared)
  • 1× TE buffer (10 mM Tris·Cl, pH 7.5, 1 mM EDTA)
  • qPCR reagent kit (Bio‐Rad, cat. no. 172‐5120)
  • Rotating mixer (Thermo Scientific)
  • Eppendorf DNA LoBind microcentrifuge tubes
  • Microcentrifuge
  • 6‐tube magnetic separation rack (e.g., NEB, cat. no. S1506)
  • Thermal cycler (e.g., Bio‐Rad CFX96 Touch Real‐Time PCR Detection System or similar)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5, Voytas, ), quantifying DNA using a spectrophotometer ( appendix 3D, Gallagher and Dejardins, ) and using a Nanodrop instrument (APPENDI , Dejardins and Conklin, )
NOTE: It is important to use sterile technique to avoid bacterial growth in reagents and environmental DNA contamination during the enrichment process.NOTE: It is also recommended that reagents be stored at ‐20°C, except Protein A beads (4°C storage), to minimize DNA contamination from environmental bacteria. Reagents stored at 4°C or room temperature (25°C) can support bacterial growth.

Alternate Protocol 1: Eluting Captured Host DNA

  Additional Materials (also see protocol 1Basic Protocol)
  • Proteinase K (800 U/ml)
  • Vortex mixer
  • Waterbath or thermomixer set to 65°C
  • Microcentrifuge

Alternate Protocol 2: Ethanol Precipitation of DNA

  Additional Materials (also see protocol 1Basic Protocol)
  • 100% ethanol

Alternate Protocol 3: Quantitation and Validation of Enrichment by Agarose Gel Electrophoresis

  • 6× Gel Loading Dye blue
  • λ DNA‐Hind III Digest
  • Agarose for large fragment analysis
  • Agarose gel electrophoresis apparatus
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Internet Resources
  Updated information on the DNA enrichment kit may be found at the above Web site.
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