Microarray Creation Workflow

1. The contigs[contigs reference] were submitted to combimatrix for probe design. Unique probes could not be created for all of the sequences[probe numbers reference] and some probes were duplicated because they were designed for contig sequences that were likely from gene families.
2. Not all of the probes could fit on the array and some probes were 'bumped'.
3. The positions of reporters on the array were determined in a semi-random process by Combimatrix's probe design software. The software provides a list of the probes that were placed on the array and a chip design file that describes the positions of probes on the array. The chip design file is used to generate the galfile.
4. Sequences are synthesized directly onto electrodes on a microchip.[footnote to combimatrix technology]

Microarray Statistics

Input ESTs to Bioinformatics
Brassica napus	659,227
Brassica oleracea	68,393
Brassica rapa	54,206
Total EST's	781,826
(estimated genome coverage	65% based on homology with Arabidopsis thaliana)

Unique Sequences from Bioinformatics
EST's Direction known	72,990
EST's Direction unknown	22,428
EST's Sequences for probe design	95,418

Control Sequences
Stratagene SpotReport(r) Alien(r) sequences (catalog # 252550)	10 (3 replicates of each) Fig.1

CombiMatrix 90K Chip - Chad - Fix these numbers
Total electrodes on chip	94,928 (272 columns x 349 rows)
Combimatrix QC electrodes	4,398 (null and negative controls) Fig. 2
Electrodes available for user probes	90,530
probes are custom synthesized on chip - not spotted or printed

CombiMatrix Bn90k-v1.1 Chip
Total electrodes on chip	94,928 (272 columns x 349 rows)
Unique IDs on the chip (including 'empty' and 'QC*')	90,565
probes are custom synthesized on chip - not spotted or printed

CombiMatrix 90K Probe Design
Sequences submitted	95,417
Probes not found	3,923 (did not meet probe design criteria)
Probes found	91,494
Duplicate probes	529 (from gene families - 94% homology threshold)
Bumped sequences	465 (not enough electrodes on chip)
Designed probes on chip	90,500

Probe Design Criteria

• Probe length = 35-40 mer
• Probe melting temperature = 70-75 C
• Sense probes for anti-sense targets (labeled cDNA)
• Anti-sense probes available by special synthesis request only
• Probes restricted to a portion of target
• Default is the 3 prime 1000 base pairs
• More detailed probe design criteria is available on request

* 90K Chip Layout (Fig 1 and 2) * Populated at random (serial and grouped layouts available by special synthesis request only) * Control probes (1 set in upper left hand corner, 2 sets populated at random)
Fig. 1 Stratagene SpotReport(r) Alien(r) Control Probes (1 rep of each in upper left corner, 2 reps of each populated at random on microarray)
Fig. 2 Quality Control Hybridization with CY5 labeled random 9 mer Combimatrix (null) quality control spots are arranged in geometric patterns that divide the microarray into 9 blocks.

Notes

1. IDs and descriptions of sequences in the contigs file
   
   • All sequences have an ID as described below followed by a description with 5 fields:
   • Singletons (contigs with one member) often have an id like this: BNAEN1GH_UP_052_H12_21JUL2006_082.ab1___PBI. This was an EST from PBI.
   • Contigs have an id like this Contig1_9_final
   • IDs and descriptions of sequences in the probes files.

V1.0 Other Files

V1.1 Other Files

• Galfile with readable ID added as an additional column This readable id is a simple serial number starting with 1 and proceeding to the end