EST assembly and Microarray development
EST assembly and microarray development.
All new sequences (454 and Sanger) will be assembled along with the current set
of approximately 340,000 Gossypium ESTs using proven techniques.
Comparisons between allotetraploid and diploid reads are used to diagnose
genome-of-origin for reads from the allotetraploids. Thus, homoeo-SNPs will be
identified by sequence comparisons between diploid ESTs and allele-SNPs will be
identified by respective pair-wise comparisons between the recurrent and donor
parents for each population. We have written software to identify homoeo- and
allele-SNPs and it has been successfully used to generate two versions of a
homoeolog-discriminating microarray for gene expression biases. Once homoeo-
and allele-SNPs have been identified, probes are designed from the flanking
sequences to specifically target the SNPs. Probe design includes using our SNP
identifying software to output flanking bases (e.g. 30 to 40 bases) surrounding
the SNP, and culling duplicates and low complexity probes. Final probe
selection at Nimblegen is based on probe content and probe length to optimize
the entire array for uniform annealing temperature. Microarray probe sets are
subjected to a final summary and review before array manufacturing.