Transcript Gene Discovery and Genome Browsing

Gene Discovery
& Genome Browsing
• Background – myself
• NsGene – DTU satellite
• Parkinson Disease (Affymetrix GeneChip)
• Analysis of fetal brain tissue
• Genome browsing
• Introduction to the UCSC BLAT browser
Background
• Thomas Nordahl Petersen
•Chemist, Ph.D protein Crystallography, University of
Copenhagen
•Computational Scientist, SBI-AT
•Prediction of protein structure, secondary structure,
fold recognition, homology modeling
•Bioinformatics - Gene discovery, NsGene
Devolop novel cell and gene based products for the
treatment of neurological diseases.
ECT Products
ECT for Parkinson’s Disease
• Growth of cells in a capsule
matrix
• The therapeutic protein be
released directly in the
relevant brain area
• Safe delivery across the
blood-brain-barrier
• Michael J. Fox foundation granted US $3 million
to support a clinical “proof-of-concept” (May 2004)
Factor Products
• Identification of novel genes by
use of bioinformatics
• Neublastin (GDNF family –
potent neuroprotective effects)
• Scanning the human genome or assembled protein sets
for different features of interest
A case study
• Search for Parkinson related gene(s)
• Affymetrix GeneChip experiments
• Fetal brain tissue
Parkinson Disease
Degenerative central nervous system (CNS) disorder
Parkinson Disease
Loss of dopamine producing brain cells
Parkinson’s Disease
• Dopamine from Substantia
nigra activates neurons in
Striatum/Basal ganglia
• Important for initiation of
movement
Cure for Parkinson’s Disease ?
Parkinson disease may be cured provided that new dopamine
producing cells replace the dead ones.
Dopamin producing brain cells from aborted foetuses have been
operated into the brain of parkinson patients and ín some cases
cured the disease. Brain tissue from approx 6 foetuses were needed.
Major ethical problems !
Search for a protein drug is the only valid option
Parkinson Disease
Dopamine producing cells
• Dopaminergic neurons can be found in the ventral
part of the mesencephalon (VM) from approximately 6
weeks
• No dopaminergic neurons can be found in the
neighbouring dorsal part (DM).
• Dopaminergic differentiation by use of GeneChips to
compare the expression profiles of VM and DM
Fetal brain tissue
Midbrain mesencephalon
+ Dopamine producing
Vm
Dm
cells
- Dopamine producing
cells
• Aborted feotus brain tissue – Karolinska hospital
• Feotus of age 6-10 weeks, 2 cases
Midbrain mesencephalon
Dm
Vm
- Dopamine
+ Dopamine
producing
cells
producing
cells
Dopamine producing cells at the interface ?
Isolate the two samples (Vm/Dm)
RNA purification + amplification
Affymetrix genechip analysis
GenePublisher
(program by Steen Knudsen)
•Scale, normalize the Affymetrix GeneChip experiments
A1
A2
A2
B1
B2
B2
P-value
319
315
314
44
48
38
1.26e-07
314
334
327
443
434
444
6.55e-05
1980
1974
1973
1801
1785
1763
6.77e-05
123
123
126
87
88
93
8.01e-05
103
101
104
77
78
73
0.000112
107
107
111
79
77
82
0.000124
128
123
117
189
184
196
0.000142
179
179
186
145
147
149
0.000191
78
77
79
86
87
87
0.000202
96
90
93
136
129
138
0.000215
Vulcano plot
P-value
Log2 Fold change
Assigning Affymetrix GeneChip probes
to a protein sequence
~20.000 probes on each of the A/B Affymetrix chips. The
probes are normally not a part of a protein sequence.
5’
Unigene sequence (cDNA)
Blast
IPI protein sequence
inferred
3’
Blast
Affymetrix probe
Internal database
Signal Peptide prediction
Conclusion – so far
• The most up-regulated genes include several ‘known’
genes like dopamine transporter (good positive control)
•The most interesting genes are the ‘unknowns’ that
were up-regulated in Vm. Futher analysis is ongoing.
• Roland JR et al., Exp Neur (2006) Vol 198,2,427-437
“Identification of novel genes regulated in the
developing human ventral mesencephalon”
BLAT
The Blast Like Alignment Tool
• Smith-Waterman (1980)
• Local alignment (best alignment)
• FASTA (1988)
• Fast alignment
• BLAST-programs (1990,1997)
• Query seq versus an indexed database
• MegaBLAST (2000)
• Fast alignment of DNA sequences
• SSAHA (2001)
• Maps sequence reads to genome
BLAT
Blast Like Alignment Tool
• BLAT (2002)
• Very fast searches (MySQL database)
• Handle introns in RNA/DNA alignments
• Data for more that 30 genomes (human, mouse, rat…)
Splice sites
Exon
Intron
Exon
BLAT genome Browser
http://genome.ucsc.edu//
BLAT genome Browser
Using a search term or position eg Chr1:10,234-11,567
BLAT genome Browser
http://genome.ucsc.edu/
BLAT genome Browser
Using a protein or DNA sequence
Blat genome Browser
BLAT genome Browser
”Details”
Blat genome Browser
BLAT genome Browser
”Browser”
BLAT genome Browser
Zoom in ’base’
BLAT genome Browser
”Browser”
Trembl
SwissProt
Hypothetical
BLAT genome Browser
”Description & Page Index”
BLAT genome Browser
”Description & Page Index”
Blat Genome Browser
”tracks”
BLAT genome Browser
”SNPs”
BLAT genome Browser
”SNPs”
Gene Sorter
Gene sorter