Genotyping by sequencing (GBS) is a simple highly-multiplexed system for constructing reduced representation libraries for the Illumina next-generation sequencing platform developed in the Buckler lab by Rob Elshire. Key components of this system are: reduced sample handling, fewer PCR and purification steps, no size fractionation and inexpensive barcoding. We use restriction enzymes to reduce genome complexity and avoid the repetitive fraction of the genome.
GBS Method Paper
Presentation on GBS
GBS Protocol Details and Notes (including pricing, dilution calculator, etc.)
Bar Coded Adapter Generator (outside link)
384 Plex ApeKI Adapters (Updated May 11, 2012 to correct two bad bar codes.)
Jesse Poland has developed a two enzyme system based on our GBS method. His paper is now available. Supporting information includes 48 and 384 plex sets of adapters for PstI.
Barcode Adapter Design:
The barcoded adapters are designed with four criteria:
(1) The barcode does not contain or recreate the enzyme cut site.
(2) Any barcode in a set is at least two substitutions away from any other barcode.
(3) They vary in length as a set (to avoid the all cut site bases appearing at the same positions in the sequencing read).
(4) They contain the complementary sticky end to the enzyme cut site.
Costs per Sample*:
* These figures include maintenance and depreciation of the sequencers as part of the sequencing costs, but not the aquisition and upkeep of other laboratory equipment.
This research is supported by the USDA-ARS and a grant from the National Science Foundation.