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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.
Quick Links:
GBS Bioinformatics
FAQ
GBS Method Paper
Presentation on GBS
96 Plex GBS Protocol
Dilution Calculator
Bar Coded Adapter Generator (outside link)
384 Plex ApeKI Adapters (WARNING: Adapters 178 and 250 are bad and are being redesigned. We will post the updated file when it is available.)
Method Overview:
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.
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