Transcript CATMA, the Complete Arabidopsis Transcript Micro Array.

CATMA, the Complete
Arabidopsis Transcriptome
Micro Array.
Jim Beynon
Horticulture Research
International
The Collaboration
P. Hilson
T. Altmann
S. Aubourg
J. Beynon - GARNet
F. Bitton
M. Caboche
M. Crowe - GARNet
P. Dehais
H. Eickhoff
E. Kuhn
S. May - GARNet
W. Nietfeld
http://jic-bioinfo.bbsrc.ac.uk/CATMA/
J. Paz-Ares
W. Rensink
P. Reymond
P. Rouzé
U. Schneider
C. Serizet
A. Tabrett - GARNet
V. Thareau
M. Trick - GARNet
G. van den Ackerveken
P. Van Hummelen
P. Weisbeek
M. Zabeau
GARNet GST Project
Complement NASC EST arrays
Add approx. 9000 GSTs
Milestones: Start
2,500 GSTs
6,000 GSTs
9,000 GSTs
April 2000
April 2001
April 2002
March 2003
HRI: Jim Beynon, Alex Tabrett
JIC: Martin Trick, Mark Crowe
AFGC Amplification project
3000 GSTs to be amplified
HRI carried out 1000 amplifications
Delivered to NASC April 2001
Remaining primers available at HRI but
superseded by CATMA project
Goals of CATMA
Produce Genome Sequence Tags (GSTs) for all
predicted genes in Arabidopsis accession Columbia
To use these GSTs to produce a complete genome
Microarray.
Predicting the genes in Arabidopsis
SplicePredictor
Netstart
genomic
fragment
RepeatMasker
NetGene2
EuGène
Blastn
genes
Blastx
EuGene Results on
Chromosome V
AGI: 5888
EuGene: 6715
Chromosome V
350
300
250
200
150
100
50
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Mb
SPADS:Specific Primer and Amplicon
Design Software
Primer3
gene sequence
exon coordinates
GST
specificity
Blastn
SPADS
GST
primer
specificity
Blastn
SPADS Results on
Chromosome V
AGI: 5888
EuGene: 6715
GSTs: 4784 (73%)
Chromosome V
350
300
250
200
150
100
50
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Mb
Distribution of GSTs lengths
3000
150-200 bp: 42%
200-300 bp: 36%
300-500 bp: 22%
2500
2000
1500
1000
500
0
150
180
210
240
270
300
330
360
390
420
450
480
bp
Position of GSTs
5’
center
CDS
ATG
5115
(24%)
3’
UTR
stop
3267
(16%)
12701
(60%)
Easy Re-amplification
16
x
24
U 5’ col. 4
U 3’ row 2
No cross contamination between wells
First round PCR with
specific primer pairs
SS5 5’ ’
Genomic BAC DNA
S3’
Second round PCR with
universal primer pairs
U5’
Primary amplicon
U3’
Current Status of CATMA
Genes identified within CATMA
SPADS has designed
GSTs as % of total genes
29,775 genes
21,420 GSTs
72%
Total Primary amplifications
% Success in processed plates
% Success after plate correction
16,280 GSTs
90.2%
96%
CATMA: The Future
SPADS has yet to design primers from 28% of
genes identified within CATMA
Extending 150bp into 3’UTR will probably yield
a further 10% of GSTs
Highly homologous genes will never yield GSTs
Design best possible and note on website
CAGE: Compendium of
Arabidopsis Gene Expression
Having constructed the CATMA arrays we need
to standardise them
We are developing a project to produce a compendium
dataset
This will be available over the Web to enable users
of CATMA arrays to relate their data to that
of others
Hughes et al. (2000) Cell 95, 717-728
Functional discovery via a compendium of expression profiles
RNAi: A key tool for future
Arabidopsis research
Insertional knockouts are limited in their use
RNAi is a complementary aproach
CATMA GSTs are ideal for RNAi
We propose to produce a seed resource with
RNAi constructs for all genes
Signal According to Length
100000
FLOWER BUD
10000
signal
1000
100
10
1
100
predicted gene GST
known gene GST
intergenic region GST
highly expressed cDNA
negative control
600
1100
1600
probe length (bp)
2100
Success of EuGene
correct partial
actual gene
gene
split missing
genes models models genes genes
238
182
(76%)
50
(21%)
5
(2%)
actual missing
exons exons
1639
51
(3%)
1
(0.5%)