General information from Affymetrix

1. Who is making the array?

A Chicken Consortium within AvianNET is working with Affymetrix to create the array.

2. How will the array be designed and what will be on it?

The primary source of content will be derived from mRNA sequences obtained from GenBank and EST sequences from dbEST. When appropriate and available, untrimmed EST sequences will be used to improve polyadenylation site and signal detection and Phred scores will be used to improve the cDNA assembly results.

A draft genome assembly of the chicken will be used to improve the design in a number of ways. Orientation of cDNA sequences is improved through analysis of consensus splice sites within genome alignments. Low quality portions of EST reads are identified through cDNA alignments to the genome. Finally, content that can be aligned to the genome is prioritized higher for inclusion on the array. It should be noted that the genome will be leveraged in a conservative manner to analyze and annotated cDNA sequences.

The subcluster assembly will be processed to generate probe selection regions just upstream of all putative polyadenylation sites. This approach identifies alternative polyadenylation sites internal to the assembly end. The probe sequences will be derived from a conservative consensus sequence generated from the subcluster assembly.

Probes and probe sets will be selected as described by the “Array Design for the GeneChip Human Genome U133 Set” technical note. In short, a thermodynamic multiple linear regression model will be used to predict probe performance. Probe sets consisting of multiple probe pairs (one perfect match and one mismatch probe) will then be selected based on predicted probe characteristics, such as performance, uniqueness metrics, and spacing rules.

3. What is an Affymetrix probe set and how are these chosen?

Gene transcripts are represented by probe sets that consist of multiple 25mer oligonucleotides. Independent measurements provided by multiple probes are required for specificity and sensitivity.

Two basic types of probe sets are created: -

• Those that represent gene sequences uniquely
• Those that represent multiple closely related gene sequences

Affymetrix probe sets typically consist of 11 to 16 probe pairs. For the Xenopus array 16 probe pairs were selected per probe set. A probe pair consists of a 25mer perfect match oligo and a reference 25mer mismatch oligo. For the eukaryotic gene expression assay, labeled antisense cRNA target is hybridized to the complementary 25mer probes.

4. How do I get information about the Affymetrix assay?

A thorough description and diagram of the assay can be found on Affymetrix.com. (http://www.affymetrix.com/support/technical/index.affx). Briefly, the starting material is either total RNA (minimum of 5 µg) or poly-A mRNA (minimum of 0.2 µg). The sample is reverse transcribed, using a T7-oligo(dT) primer and double-stranded cDNA is synthesized. The double-stranded cDNA, with the incorporated T7 promoter, is then used as a template in the subsequent in vitro transcription reaction. Biotinylated UTP and CTP are incorporated into cRNA during the in vitro transcription reaction. Labeled cRNA is detected via streptavidin-phycoerythrin staining.

5. Where (How) do I run my experiments?

Affymetrix arrays must be processed on GeneChip® Systems (processing is also available in institutional core labs, or service providers for those who do not have systems installed locally in department or individual labs).

6. Are annotations and support available?

Annotations will be available via the public NetAffx database at Affymetrix.com. Annotations will be updated on roughly a quarterly basis and will include at minimum: probe sequences, consensus sequences, and annotation in a tab-delimited file using information form many sources, in particular the chicken-Ensembl database planned for 2004.

7. When will it be ready?

The goal is to have a GeneChip® probe arrays available by the second half of 2004.

8. How much will it cost per chip and per experiment?

Chip prices are volume-based and are set based on institutional agreements. Academic pricing will honor local institutional agreements, and the number of GeneChips that will constitute a minimum order is 5. Please check with your local Affymetrix representative who can assist with more accurate pricing.

A general comment on the cost per experiment: In almost all cases, the biological variation is substantially larger than the variation caused by chip itself. To determine what level of variation there is, a pilot experiment is recommend consisting of 3 controls and 3 treatments to determine how much statistical power is required.