title: Agilent-022854 Custom Eucalyptus grandis expression array V1
status: Public on Oct 06 2012
contact.city: Bet-Dagan
contact.first: Ron
contact.last: Ophir
contact.address: POB 6
contact.department: Fruit Tree Sciences
contact.country: Israel
contact.institute: Agricultire Research Organization
organism: Eucalyptus grandis
manufacture_protocol: Expression array was designed based on 71,208 contigs published previously (Novaes, et al. 2008). Initially, we validated that all contigs did not retain “adaptor” and “vector” sequences by shell and perl scripts. We selected 24,264  long contigs (N90 =101bp) and then set the sequence orientation (sense/antisense) by running the contigs against public databases (e.g., NR (http://www.ncbi.nlm.nih.gov/), Arabidopsis (http://www.arabidopsis.org/), proteins of other plant at TAIR (http://www.arabidopsis.org/), PlantGDB (http://www.plantgdb.org) and UniRef (http://www.ebi.ac.uk/) at EBI). We filtered out for high fidelity sequences based on e-value (<1e-5) and query coverage (>85%) at least in one database or with less than 85% query coverage, but in the same orientation in all mentioned databases. The orientation of contigs that had no significant hit was defined by whether they had poly-A or poly-T. If poly-A/poly-T sequences were not detected, we set the orientation contig by open reading frame (ORF) based on the criterion that the length ratio of the forward to reverse complementary sequence ORF is greater than 10.  Probes were designed between 56 to 60 base pairs long and Tm was optimized by using OligoWIZ (Wernersson and Nielsen 2005). In addition, low complexity cross hybridization was avoided and a high priority was given to probes at the 3’ position.