The original sequences were generated using dideoxy shotgun sequencing and clone walking of plasmid and BAC clones using paired-end information for assembly purposes, and sequencing of PCR products to close gaps.
The sequencing of the colony PCR products was performed using dideoxy chain-termination sequencing on an Applied Biosystems ABI3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA).
The standard approach to establish genomics resources for non-model plant species involves the random generation of expressed sequence tags (ESTs) from a cDNA phagemid library using dideoxy chain-termination (Sanger) sequencing technology.
In parallel, 9,578 reads were generated from a library constructed from the same normalized cDNA population using dideoxy chain termination Sanger sequencing, which upon clustering and assembly generated 4,689 unique sequences.
Plasmid DNA for each genomic locus was sequenced using dideoxy chain termination method with ABI 3730XL 96-capillary sequencer.
For DNA sequencing, plasmid DNAs prepared with a commercial column (Qiagen) served as templates in cycle sequencing reactions performed using dideoxy chain termination (Sanger et al, 1977) Sequence were analysed using fluorescence DNA sequencers.
DNA was sequenced using dideoxy-terminator cycle sequencing technology.
PCR products were purified and both heavy and light strands were sequenced using dideoxy chain termination chemistry with Big Dye v3.1 following recommended ABI protocols and run on an ABI3100 automated capillary sequencer.
Bi-directional DNA sequencing analysis performed by using dideoxy chain termination method with a T7 DNA polymerase sequencing kit (Pharmacia Biotech Inc., Piscataway, NJ, USA), or directly sequenced using a Taq dideoxy terminator cycle sequencing kit on an ABI 377 DNA sequencer (Perkin-Elmer, Foster City, CA, USA).
PCR products were sequenced at the Australian Genome Research Facility AGRFF, Brisbane, Australia) using dideoxy dye chain-termination technology (Applied Biosystems).
Variants that failed genotyping using Sequenom assays were typed in family and unrelated controls using bidirection dideoxy sequencing of amplified products or Taqman genotyping assays-by-design run on a 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
