T. gondii GBrowse Ancillary SiteThe ToxoGeneChip
ToxoDB Home Final Design SNP Discovery Host Profiling ChIP Genetic Markers EST based SNPs Transgene Reporters Library Files P. falciparum SNPs

Ordering Instructions:
Affymetrix T. gondii GeneChips are distributed by the Penn Microarray
Facility for $175 per chip plus shipping.  Payment may be made by purchase
order and institutional check, credit card, or bank wire transfer.  To order
GeneChips, you must 1) set up an account, and 2) place your order(s).  Both
steps can be completed by visiting www.med.upenn.edu/bmcrc.  First, click on
the Guide to Requesting Service for instructions on setting up an account.
When you receive account confirmation, choose Microarray from the list of
Resource Laboratories and follow Request Other Services.

Copy of email sent to the Toxoplasma community describing the array design:
Dear colleagues:

I am writing to detail arrangements that have been made for the development and distribution of photolithographic microarrays 
for the Toxoplasma gondii research community.  The bottom line is this: arrays suitable for whole-genome expression profiling 
and genotyping of T. gondii are expected to be available in May 2006, at a cost of $175/chip (plus shipping).  Please let me 
know if you require a hard copy of any of the following information (e.g. for grant support, etc).  
Needless to say, you should also feel free to forward this e-mail to others.

As you know, a complete reference genome is now available for T. gondii strain ME49 (type), with sequences for the RH (type I) 
and VEG (type III) strains to follow shortly.  These sequences, along with whole genome annotation of gene models, and mapped 
genetic markers, ESTs, and SAGE tags, are accessible at http://ToxoDB.org.

The availability of complete genome sequence, annotated gene models, and SNPs identified from genome mapping and EST sequencing 
projects makes possible a wide variety of hybridization array-based studies, and Computational Biology PhD student Amit Bahl has 
worked with David Kulp (UMass, Amherst), Eric Schell and Gene Tanimoto (Affymetrix), and several members of the Toxoplasma 
research community to design a single photolithographic array that permits a several analyses (although not necessarily in the 
same experiment).  See below for information on how to visualize the available probes ... it may still be possible to incorporate 
any urgent requests for revision, but only if these are received before the end of this week!

One primary application for the microarray is to provide whole genome expression profiling, using at least 11 perfect match probes 
for each of the ~8000 predicted genes in the T. gondii genome (including both the apicoplast and mitochondrial genomes).  We have 
also incorporated probes for common reporter genes (CAT, LUC, GFP, etc), and several hundred human and mouse genes (and a few from 
cat) -- including various controls (actin, HGPRT, etc), immune effector molecules (cytokines, receptors, etc), and genes whose 
expression is suspected from previous microarray studies to be altered by infection.  Config files detailing these probes will
permit analysis of gene expression profiles using a variety of publicly and commercially available algorithms.

These microarrays may also be employed for genotyping, using standard software from Affymetrix to analyze highly redundant 
allele-specific probes (40/SNP) for 228 mapped polymorphisms distributed throughout the T. gondii genome.  We have also incorporated 
less redundant probes (4/SNP) for an additional 3312 strain-specific polymorphisms known from EST sequencing, and will develop 
independent software for analysis of these probes, providing median genome-wide mapping resolution of ~4kb.  In addition, the 
T. gondii arrays also include a probes for SNP detection in several highly polymorphic genes, including SAG1/2/3/4, SRS1/2/9, 
GRA3/6/7, ROP1/16, AMA1, MIC2, B1, BSR4, and a few intronic sequences from other genes.

Finally, some probes are intended to facilitate gene finding and gene model validation; to provide pilot-scale (i.e. not genome-wide) 
studies on antisense expression, chromatin immunoprecipitation, etc; and to address a variety computational and statistical questions 
(gene model validation, background levels, etc).

We have utilized the Affymetrix 169 format for these arrays, which requires a relatively high up-front design fee, but will enable us 
to provide chips to the community at a relatively low cost per array.  At present, we are projecting a cost of $175/chip + shipping.  
The Penn Microarray Facility has agreed to coordinate distribution, without additional charge.  The completed designs are now at 
Affymetrix for final review and approval, and we are expecting chips in May 2006.

How to browse Toxoplasma gondii chip probes.  All Toxoplasma probes have been mapped to the T. gondii genome, and may be reviewed at
http://ancillary.toxodb.org/cgi-bin/gbrowse/ancillary/.  Note that it may take a few minutes for this page to load.

We would like to encourage anyone who has helped with the chip design, or who plans to  utilize this array to take a careful look
at  this web site, in order to familiarize yourself with the array and to help us review the design.  It may still be possible to 
incorporate any urgent requests for revision, but only if these are received before the end of *this* week ... once Affymetrix 
etches the array masks, they cannot be changed!

Clicking on the above link should take you to a view of 110 kb near the right telomere of chromosome VIIb (if not, click on the 
first example shown).  Reading from top to bottom, this view shows:

Genetic markers (green for type I, distinguishing RH from ME49 and VEG, blue = type II, red = type III)
- characterized genetic markers from the Sibley lab's NAR paper (none in this segment)
- putative SNPs based on EST analysis (... as indicated by mouse-over)

Genotyping probes
- for characterized markers (40/SNP)
- for EST-based SNPs (4/SNP)

For further information on genetic markers and EST-based SNPs, see Genetic Markers and EST based SNPs links in the header 
(Note to Sibley and Boothroyd labs: please spot check sequences).

Genes (red vs blue indicates strand)
- annotated genes (mouse-over for more detailed information)
- BLAST hits
- EST alignments
- SAGE tags

Expression profiling probes
- for annotated genes (11 probes near the predicted 3' end of each gene)
- noncoding genes (none in this region)
- transcript discovery probes based on BLAST hits outside of annotated genes
- transcript discovery probes based on outside of annotated genes
- transcript discovery probes based on TBLASTN hits outside of annotated genes
- additional exon, intron and antisense probes (none here; available for chromosome 1b only)
- ChIP probes (none here; pilot study for a few selected regions only)
- resequencing probes (densely tiled probes for a few selected genes, such as AMA1 (55.m00005), shown here)

Note that for each annotated gene, there are expression profiling probes in the "Nuclear Coding Probes" track.  Many genes have 
BLAST or EST support, but there are regions with such hits and no annotated genes.  For the most promising of these regions, 
we've tiled extra probes as seen in the various "Transcript Discovery" tracks.  We've also densely tiled select surface antigens, 
such as AMA1) seen in this view.  These probes can be seen in the Resequencing Probes track and further details on tiled antigens 
in the header at SNP DISCOVERY.  For details on all design choices, see FINAL DESIGN in the header.

Now click on the second example, Ib:1..50000.  In addition to expression profiling probes, note additional probes for each exon, 
intron, and antisense region for genes on chromosome Ib - these can be seen in their respective tracks.

A third example (Chromosome:Ib) displays all of Chromosome Ib, permitting visualization of haplotype structure.  Note that in 
order to permit this page to load, all annotation and expression profiling probe tracks have been turned off (you can modify 
this yourself using the "Tracks" menu at bottom.

There are also many other examples that you can explore, covering most of the genomic features that we've included in our 
design ... and of course you should feel free to explore on your own!  In general you can jump to any genomic location of 
interest by filling in the "Landmark or Region" box (use format :..).  Please do check your favorite 
genes/regions to see if we have provided the required probe coverage.  If not, please e-mail Amit (abahl@mail.med.upenn.edu) 
with details immediately!

Sincerely, David S. Roos

P.S. Among the many members of the Toxoplasma research community who have assisted with chip design, Jim Ajioka, John Boothroyd, 
Jon Boyle, Asis Khan and David Sibley helped to define probes for genetic analysis (including EST-based SNPs); Boothroyd, Boyle, 
Sibley and Chris Hunter provided suggestions for host cell markers; Jean Feagin provided feedback on noncoding RNAs; Ali Hakimi 
and Bill Sullivan on promoter analysis; and Feng Chen, Zhongqiang Chen, Aaron Mackey, Lucia Peixoto, and other members of the 
Roos lab provided additional assistance.