Genotyping‐by‐Sequencing

Jason G. Wallace1, Sharon E. Mitchell2

1 The University of Georgia, Department of Crop and Soil Sciences, Athens, 2 Cornell University, Genomic Diversity Facility, Ithaca, New York
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20042
Online Posting Date:  March, 2017
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Abstract

Genotyping‐by‐sequencing (GBS) refers to a suite of related methods that obtain genotype data from samples by using restriction enzyme digestion followed by high‐throughput sequencing. GBS is a refinement of restriction site–associated DNA sequencing (RADseq) methods, with a goal of being able to perform library preparations quickly, cost‐effectively, and in a high‐throughput manner. This protocol contains the steps necessary to go from purified DNA to Illumina‐ready libraries. It also covers the considerations that go into planning a GBS experiment. © 2017 by John Wiley & Sons, Inc.

Keywords: GBS; genotyping; high‐throughput sequencing; RADseq; restriction enzyme

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: One‐Enzyme Genotyping‐by‐Sequencing
  • Alternate Protocol 1: Two‐Enzyme Restriction Digestion
  • Support Protocol 1: Preparation of Adapters and Concentrated Stock Plate
  • Support Protocol 2: Optimization of Adapter Concentration
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: One‐Enzyme Genotyping‐by‐Sequencing

  Materials
  • Concentrated adapter stock plate at 3 ng/μl (see protocol 3)
  • Purified sample DNAs (30 ng/μl) in 96‐well plates
  • Restriction enzyme and 10× restriction enzyme buffer
  • HPLC‐grade, sterile water
  • T4 DNA ligase and 10× T4 DNA ligase buffer with ATP
  • Commercial column‐based PCR cleanup kit (e.g., Qiagen QIAquick PCR Purification Kit or equivalent)
  • Taq polymerase master mix
  • PCR Primer 1 and Primer 2 stocks (50 μM) in 1× TE or water
  • 1× Tris‐EDTA (TE) buffer: 10 mM Tris·Cl (pH 8.0)/1 mM EDTA
  • Dilution buffer: 10 mM Tris·Cl (pH 8.0)/0.1% Tween20
  • 96‐well PCR plates
  • Air‐permeable plate‐sealing tape
  • Centrifuge with plate adapters
  • Laminar‐flow hood or SpeedVac
  • 15‐ml conical tubes
  • Thermal cycler
  • 1.5‐ml microcentrifuge tubes
  • Capillary instrument (Experion or BioAnalyzer)
  • Plate‐sealing foil
  • Vortex mixer

Alternate Protocol 1: Two‐Enzyme Restriction Digestion

  Materials
  • 200 μM separate oligonucleotide stocks of all adapter components (top and bottom strand oligonucleotides for the common adapter and all barcoded adapters) in TE buffer
  • 1× Tris‐EDTA (TE) buffer: 10 mM Tris·Cl, pH 8.0/1 mM EDTA
  • 0.2‐ml PCR tubes
  • 96‐well PCR plates
  • Plate‐sealing foil sheets
  • Thermal cycler
  • 96‐well deep‐well (at least 1.1 ml) plate
  • Vortex mixer
  • Centrifuge(s) with plate adapter
  • 1.5‐ml centrifuge tube
  • Equipment to quantify DNA (via absorption, intercalating dye, etc.)

Support Protocol 1: Preparation of Adapters and Concentrated Stock Plate

  Materials
  • 100 ng/μl high‐quality genomic DNA from species of interest (DNA sample can be from single or pooled individuals)
  • Restriction enzyme(s) of choice and appropriate buffer(s)
  • One set of adapters (both common and barcoded) at working concentrations (see protocol 1Basic Protocol, step 1; default working concentration is 0.3 ng/μl per adapter, or 0.6 ng/μl for the pair)
  • 10× T4 DNA ligase buffer with ATP
  • T4 DNA ligase
  • Molecular biology‐grade water
  • Column‐based PCR purification kit (e.g., Qiagen QIAquick PCR Purification Kit or equivalent)
  • PCR Primers 1 and 2 (50 μM)
  • Taq DNA master mix
  • 0.2‐ml PCR tubes
  • Centrifuge
  • Thermal cycler
  • High‐sensitivity electrophoresis platform (e.g., BioRad Experion or Sage Science BluePippin)
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Figures

Videos

Literature Cited

  Andrews, K.R., Good, J.M., Miller, M.R., Luikart, G., and Hohenlohe, P.A. 2016. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat. Rev. Genet. 17:81‐92. doi: 10.1038/nrg.2015.28.
  Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler, E.S., and Mitchell, S.E. 2011. A robust, simple genotyping‐by‐sequencing (GBS) approach for high diversity species. PLoS One 6:e19379. doi: 10.1371/journal.pone.0019379.
  Miller, M.R., Dunham, J.P., Amores, A., Cresko, W.A., and Johnson, E.A. 2007. Rapid and cost‐effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers. Genome Res. 17:240‐248. doi: 10.1101/gr.5681207.
  Poland, J.A., Brown, P.J., Sorrells, M.E., and Jannink, J.L. 2012. Development of high‐density genetic maps for barley and wheat using a novel two‐enzyme genotyping‐by‐sequencing approach. PLoS One 7:e32253. doi: 10.1371/journal.pone.0032253.
  Rogers, S.O. and Bendick, A.J. 1994. Extraction of total cellular DNA from plants, algae and fungi. In Plant Molecular Biology Manual (S.B. Gelvin and R.A. Schilperoort, eds.) pp. 183‐190. Springer, The Netherlands.
  Torkamaneh, D., Laroche, J., and Belzile, F. 2016. Genome‐wide SNP calling from genotyping by sequencing (GBS) data: A comparison of seven pipelines and two sequencing technologies. PLoS One 11:e0161333. doi: 10.1371/journal.pone.0161333.
Key References
  Elshire et al., 2011. See above.
  Original GBS protocol.
  Poland et al., 2012. See above.
  Two‐enzyme GBS.
  Andrews et al., 2016. See above.
  Review of various restriction site‐associated DNA sequencing methods (including GBS), with pros and cons of each.
  Torkamaneh et al., 2016. See above.
  Comparison of GBS bioinformatics pipelines with and without a reference genome.
Internet Resources
  https://bitbucket.org/jerlar73/fastgbs
  Fast‐GBS: Software for GBS.
  http://catchenlab.life.illinois.edu/stacks/
  STACKS: Software for GBS.
  http://www.maizegenetics.net/tassel
  TASSEL: Software for GBS.
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