Transcript Level Expression Profiling from Predicted and Transcribed

Transcript Level Expression Profiling from Predicted and Transcribed Sequences with a 5 µm, PM-only Tomato Array Affymetrix Confidential

Expression array design concepts 3’ IVT Probe-set (Traditional strategy) Single probe-set per gene (if possible) Complicated by alternative polyadenylation and splicing WT with every predicted Exon (Exon Array strategy) Select equal number of probes from every exon WT for Expressed Sequence or Gene Model (Gene Level strategy) Select equal number of probes from every transcript or gene coding sequence (20 -30 PM probes per gene)

Tomato transcript level expression array Benefits § Enhanced detection of ESTs, gene fragments, and SNPs § Increased genome coverage to >35, 000 transcripts through smaller feature size § Reduced array price through smaller format size § Leverage Affymetrix expertise in array and assay development

Benefits Enhanced Detection § Enhanced detection of non-3’ anchored ESTs and gene fragments with Whole Transcript Assay (WTA) – Random-primed amplification rather than 3’ anchored (oligo -d. T priming) § Advantages – Greater number of probes per transcript (≥ 30) § Enables detection along length of transcript, not just 3’ end – Good sensitivity pairing WTA with gene level array § Potential for improvement with r. RNA depletion

Benefits Increased content § TOM 1 – 12, 000 spotted c. DNAs § TOM 2 – 11, 000 spotted oligos § Affymetrix Gene. Chip Tomato Genome array – 222, 000 probes representing 9, 254 transcripts § 11 probe pair 3’ IVT strategy § Proposed Gene. Chip transcript level design – – 2. 6 million probes representing more than 35, 000 transcripts Full Unigene content § 30 or more probes per transcript • Facilitates SFP detection § 5 µm feature size § Additional space to enable other applications • Transcript discovery • Ch. IP-Ch. IP

Benefits Reduced price § No Design Fee and reduced array price – – – First 500 arrays at € 200 per array Second 500 arrays at € 150 per array All remaining orders at € 125 per array § Initial higher per array price to cover design and investment costs § Proposed Gene. Chip transcript level design – 2. 6 million probes representing more than 35, 000 transcripts

Logistics § Tomato community designate to provide sequence files of transcripts to be detected on array – Recommend sequences be annotated prior to submission § Affymetrix QCs sequences, selects array probes, and creates array design § Design review by community designate – No manufacturing until all agree on final design § Timeline for array delivery is 8 weeks once design is complete

Model Organism / Consortia Designs Experience making arrays § Arabidopsis § Grape § Tomato § Pseudomonas § Soybean § Rhesus monkey § Barley § Wheat § Xenopus § Rice § Medicago (legume, barrel cress) § Zebrafish § Corn § Canine 2. 0 § Canine § Pig § Citrus § Anopheles/Plasmodiu m § Cow § Poplar § Chicken § Cotton § Drosophila § Sugar Cane § E. coli 2. 0 § Yeast