Overview
Support
Design
Controls
Access
Experimentation
Additional Information

Overview

A consortium of researchers studying C. neoformans has arranged for the Genome Sequencing Center at Washington University to produce and distribute microarrays for interested investigators. The microarrays contain long oligomers (70-mers) that are designed to uniquely represent each gene in C. neoformans serotype D (one oligo per gene; see section on design for more information). These oligomers are arrayed on treated glass slides using split pin technology, together with oligomers representing a selection of host organisms and control oligos that represent unique A. thaliana genes (see below). The next phase of this project will consist of revision of this set to add oligos representing any serotype A genes that do not hybridize to these oligos; this will occur once annotation of that serotype is improved.

Support

This effort has been coordinated by Christina Hull (cmhull@wisc.edu), Jenny Lodge (lodgejk@SLU.EDU), and Tamara Doering (doering@wustl.edu) and is supported by The Burroughs Wellcome Fund and the following investigators (listed alphabetically): Andy Alspaugh, Maurizio del Poeta, Tamara Doering, Joe Heitman, Christina Hull, Guilhem Janbon, Jim Kronstad, June Kwon-Chung, Jenny Lodge, John Perfect, Brian Wickes, and Peter Williamson.

Design

The current set represents an effort to generate an array useful for all of the currently predicted genes in serotype D. This set includes all the C. neoformans genes that were manually annotated by The Institute for Genome Research using a combination of automated gene prediction, homology, and EST data (Loftus et al, 2005). The set also includes all genes predicted by the TWINSCAN software (Korf, Flicek, Duan, and Brent, 2001; Tenney, Brown, Vasquez, Lodge, Doering, and Brent, 2004) that are not already included in the TIGR set. The gene list generated from these two sources was used to choose a set of oligos for each gene using ArrayOligoSelector, and the final oligo for each gene was chosen to primarily optimize homology to genes predicted in serotype A, and secondarily by proximity to the 3' end of the coding sequence. Oligonucleotide sequences, sources, physical characteristics, and TIGR annotations (where applicable) are in a spreadsheet linked under Array Specific Information at the left. The TWINSCAN database used in this work may be similarly accessed.

Design of the oligonucleotide set was coordinated by Tamara Doering with scientific planning in conjunction with Michael Brent, and was executed by David Shteynberg. Helpful design suggestions were contributed by Jenny Lodge and other members of the consortium, independent checks of the oligo set were performed by Jason Stajich, and model organism sequences were assembled by Maureen Donlin. Questions about the design should be addressed to Tamara Doering (doering@wustl.edu).

Controls

Controls on the array include sequences from model organisms including human, mouse, rat, rabbit, D. melanogaster, C. elegans, Dictyostelium and A. castellanii. These sequences are included on the full oligo list above and are intended to help investigators evaluate possible contamination from host organisms in preparations of cryptococcal RNA. Most of these had background signal in experiments with cryptococcal RNA, but exceptions in some tests included oligos 5 and 13, which were slightly above background, and oligo 10 which gives a strong signal (see gene list for identification). The oligos themselves have been tested and found to have low homology to mammalian gene sequences, but it is the responsibility of the individual researcher to confirm the specificity of any signals of interest.

Additional controls include ten Arabidopsis thaliana sequences. Commercially available RNA samples (Stratagene) that correspond to each of these elements may be added to samples as positive controls. See the Array Specific Information link for more details.

Initial 'Quality control' experiments to test the array were performed by the Washington University Genome Sequencing Center, and the results are available on this site via the corresponding link. These experiments included:

• hybridization of genomic DNA of JEC21 against itself, to assess variation within experiments and the fraction of probes that hybridize (note that probe sequences that span introns may not hybridize to genomic DNA)
• hybridization of genomic DNA of JEC21 versus H99, to assess relative utility of the array for the two serotypes
• hybridization of RNA from cells grown at two temperatures, chosen because they are known to elicit different gene expression (Kronstad reference)
• hybridization of control mixtures of PCR products of C. neoformans genes to test gene list identifications

Access

Slides are available to all academic users through this web site at a cost of .50 for a slide on which the array is printed twice. This cost includes the slide and the costs of spotting (reagents and labor); users will be asked to provide FedEx account information for shipping. The oligonucleotides were purchased by the above listed consortium as a service to the academic research community. Non-academic users please contact Christina Hull (cmhull@wisc.edu) for inquiries about availability.

There is a limit to each user of 40 slides per month, please contact Tamara Doering (doering@wustl.edu) to inquire about exceptions to this limit.

To order arrays please use the Order Form link at the left. Slides can also be obtained from the microarray facility at Duke University Medical School; contact Joe Heitman (heitm001@duke.edu) for details.

Experimentation

All experimental use of these slides and data interpretation is the responsibility of the individual researcher, although some protocols are available via the link at the left.

The Microarray Facility of the Washington University Genome Sequencing Center does perform experiments on a fee for service basis to outside users. For information and pricing on these services please contact the director of the facility, Seth Crosby, at scrosby@watson.wustl.edu.

Additional Information

Maureen Donlin (St. Louis University) has performed some GO annotation on the cryptococcal microarray probes. Her description of how this was done, as well as the Excel files for those annotations, may be found at : http://genome.slu.edu/GOannotation.html, or you can follow a link from her more general cryptococcus page at http://genome.slu.edu/crypto.html.