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University of Wisconsin–Madison

Bioinformatics Resource Center

Genotype By Sequencing Analysis

The UW-Madison Biotechnology Center is currently open and following the university's COVID-19 Response regarding campus operations.

Introduction:

Genotype by Sequencing (GBS): is a molecular technique that allows genomewide genotyping of a population of organisms by performing reduced representation sequencing of their genome. GBS is a robust, simple and affordable means of SNP discovery and mapping. Users of GBS often aim to find Single Nucleotide Polymorphisms (SNPs) within a population to: promote understanding of the relationships between the organisms within, or utilize the genotyping information for genomic selection or trait association analysis.

Sample processing:

GBS analysis is traditionally performed with large batches of samples (>=96) and the goal of determining variation within the population at a sub-selected number of sites across the genome. To reduce the genome complexity we use restriction enzymes that cleave the genomic DNA with high specificity. User beware, the choice of enzyme determines the DNA to be sequenced downstream. After cleavage the DNA is purified, adapters are ligated, a DNA sequencing library is prepared and size selected before going onto the sequencer.

Sequencing is performed using an Illumina NovaSeq 6000 to produce paired end reads (2x150 bp). Data is multiplexed en mass via sample specific barcodes that will be provided to you. If you intend to demultiplex using both reads be aware that barcodes are only present on the 5' end of Read 1 (R1, forward) which is linked to a non-barcoded Read 2 (R2, reverse) via fastq specifications.

Organisms we have analyzed GBS for: 

The BRC has already analyzed GBS data for a wide variety of model and non-model organisms including:

  • Corn (Zea Mays)
  • Potato (Solanum species)
  • Sorghum
  • Carrot (Daucus Carota)
  • Ground Squirrel (Ictidomys)
  • Switchgrass (Panicum virgatum)
  • Alfalfa (Medicago)
  • Cannabis Sativa
  • Spinach (Spinacia oleracea)
  • Soybean (Glycine max)
  • Grape (Vitis vinifera)
  • Petunia (Petunia axillaris)
  • Table Beet (Beta vulgaris)
and many more...

 

Analysis Methods:

We use the most contemporary algorithms currently available for analysis of your GBS data. These include SNP calling for your entire population via the Tassel or Stacks GBS analysis pipelines. In addition to the standard pipeline outputs we provide a customized html report containing: plate, sample and variant discovery statistics combined with population structure analyses (PCA & Phylogeny).  

Example Output Report: 

Click the link to be redirected to our: Example GBS Report

Sequencing and Analysis Guidelines:

How to choose your restriction enzyme:

  • Based on previous experience
  • the GBS literature for your organism 
  • A pilot study is recommended for untested populations

GBS Sequencing Depth in a eukaryotic crop genome:

  • Sequence each sample to a depth of 1 - 5 x 106 reads/sample
  • Per GBS cut site GBS pipelines require ~5 reads/sample to call a genotype and/or variant

VCF filtering:

See our description in FAQ...

GBS Pricing Estimates:

Will you be paying for the analysis with University of Wisconsin associated funds?

How many samples will we be analyzing? (1-10000)

Is the genome your using new, i.e. will we need to configure files for it?

 

Estimated Analysis Cost

 

The Bioinformatic Resource Center is 100% cost recovery. We are able to provide free consultations as a result of the other core facilities hiring us to catch any design problems that could lead to mistakes or wasted time. Please be respectful of this time so we can keep all of our service rates low.