Transcript Slide 1

Our expertise at your demand
[email protected]
GenXPro GmbH, Frankfurt am Main
www.genxpro.de
Our Service Portfolio
- Digital Gene Expression Service:
from cells/tissues to annotated/BLASTed libraries in one to
three month
-Normalization of cDNA, sequencing and assembly
- RNA seq, microRNAs
- Taq-Man assays, Real-Time PCR service
- Identification of SNPs, molecular (genetic) markers
- Copy number variations (CNVs)
- Epigenetics
Transcriptome Analysis & Gene Discovery
SuperTag Digital Gene Expression Profiling
(ST-DGE)
An Improved version of SuperSAGE, applying second
generation sequencing and a bias free PCR technology
for optimal tag-to-gene association and quantification.
Digital Gene expression Profiling
Principle
What Gene is expressed and how often ?
Anchoring Enzyme
5’
3’
5’
3’
5’
3’
5’
3’
Streptavidin-Beads
Tagging Enzyme
cDNA
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
cDNA
AAAAAAA-3’
TTTTTTT-5’
cDNA
cDNA
AAAAAAA-3’
TTTTTTT-5’
Sequencing of
Millions of 26 bp
SuperTags
Counting, BLAST
Digital Gene expression Profiling
Principle
Streptavidin-Beads
1.Digestion with Anchoring Enzyme
5’
3’
5’
3’
5’
3’
5’
3’
cDNA
cDNA
cDNA
cDNA
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
Digital Gene Expression Profiling
Principle
What Gene is expressed and how often ?
Streptavidin-Beads
1.Digestion with Anchoring Enzyme
5’
3’
5’
3’
5’
3’
5’
3’
cDNA
cDNA
cDNA
cDNA
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
Digital Gene Expression Profiling
Principle
What Gene is expressed and how often ?
Streptavidin-Beads
1.Digestion with Anchoring Enzyme
2. First Linker Ligation
Linker 1
cDNA
3. Digestion with Tagging Enzyme
4. Recovery of Linker-Tags
Linker 1
Linker 1
Linker 1
cDNA
cDNA
cDNA
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
Highly specific 26bp “SuperTags“
Digital Gene Expression Profiling
Principle
What Gene is expressed and how often ?
Streptavidin-Beads
1.Digestion with Anchoring Enzyme
2. First Linker Ligation
Linker 1
Linker 2
Linker 1
Linker 2
Linker 1
Linker 2
Linker 1
Linker 2
3. Digestion with Tagging Enzyme
4. Recovery of Linker-Tags
5. Second Linker Ligation
5. PCR
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
Digital Gene Expression Profiling
Principle
What Gene is expressed and how often ?
Streptavidin-Beads
1.Digestion with Anchoring Enzyme
2. First Linker Ligation
Linker 1
Linker 1
Linker 1
AAAAAAA-3’
Linker 2
TTTTTTT-5’
Linker 2
Linker 2
Linker
Linker1 1
Linker 1
Linker
Linker2 2
Linker 2
3. Digestion with Tagging Enzyme
4. Recovery of Linker-Tags
5. Second Linker Ligation
5. PCR
6. Next-Generation Sequencing
7. Counting of Tags, Bioinformatics
AAAAAAA-3’
TTTTTTT-5’
Sequencing of Millions of Tags
Linker 1
Linker 2
Linker 1
Linker 2
Linker 1
Linker 2
Linker 1
Linker 1
Linker 1
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
Linker 2
TTTTTTT-5’
Linker 2
Linker 2
Counting, BLAST
Digital Gene expression Profiling
Principle
Anchoring Enzyme
5’
3’
5’
3’
5’
3’
5’
3’
Streptavidin-Beads
Tagging Enzyme
cDNA
AAAAAAA-3’
TTTTTTT-5’
AAAAAAA-3’
TTTTTTT-5’
cDNA
AAAAAAA-3’
TTTTTTT-5’
cDNA
cDNA
AAAAAAA-3’
TTTTTTT-5’
Sequencing of
Millions of 26 bp
SuperTags
Counting, BLAST
Digital Gene Expression Profiling
Quality
Quality of digital gene expression data depends on:
1. Quality of the Tag (what gene is expressed?)
2. Quantity of the Tags (how often is the gene expressed?)
Tag-Quality
The Tagging Enzyme determines Quality of Tags:
LongSAGE, other DGE platforms
18-20 bp
MmeI:
5’- GGGACNNNNNNNNNNNNNNNNNNNN -3’
3’- CCCTGNNNNNNNNNNNNNNNNNN -5’
SuperSAGE, SuperTAG-DGE
EcoP15I :
26 bp (=SuperTAG)
5’-CAGCAGNNNNNNNNNNNNNNNNNNNNNNNN -3’
3’-GTCGTCNNNNNNNNNNNNNNNNNNNNNNNNNN -5’
Tag Quality
What gene?
SuperTAGs allow unequivocal Identification
of the corresponding Gene
Enzyme
Platform
Tag-Size
e-value
BsmFI-Tag
SAGE
14 bp
105
MmeI-Tag
LongSAGE, Other platforms
18-20 bp
0,34
EcoP15I-Tag
SuperSAGE, SuperTAG
26 bp
0,00002
Tag Quality
Advantages of the SuperTAG
20 bp versus 26 bp
18-20bp (MmeI, LongSAGE)
26 bp (Ecop15I, SuperTAG)
BLAST-Hit , Mus musculus, Score = 52
CATGGTGGCTCACAACCATC CATAAC
Immunoglobulin kappa chain complex
CATGGTGGCTCACAACCATC CGTAAT
Tumor necrosis factor (ligand) superfamily, member 10 ?!
CATGGTGGCTCACAACCATC TGTAGA
Homeodomain leucine zipper-encoding gene
?!
CATGGTGGCTCACAACCATC TGTATC
Mannose phosphate isomerase 1, transcript variant 4
?!
Only the 26 bp tag can differentiate between the transcripts !
?!
Problem of PCR-introduced BIAS
Certain tags are preferentially amplified during PCR
biased quantification
The Solution: GenXPro’s bias-proof adapters (patent pending)
secure quantification
Downstream applications &
Advantages of the SuperTAG
26 bp SuperTAGs can:
• Directly be used as highly specific primer for PCR
3‘- and 5‘- RACE, in vitro PCR, qRT-PCR: new
genes & non-model organisms can be analyzed.
• Serve as specific probes: identification of genomic
or cDNA clones
• Be directly spotted on a microarray for HT analysis1
• Be used for the simultaneous analysis of two or
more organisms (pathogen/host)2
1. Matsumura et al. (2006) Nature Methods 3:469-474
2. Matsumura et al. (2003) PNAS 100: 15718-15723
Digital Gene Expression vs. Microarrays
Major Advantages of SuperTAG-DGE versus Microarrays
• No false positives, no cross hybridisation
• Open architecture platform: any gene detected, novel
genes, unexpected transcripts, antisense transcripts
• Reliable quantification of the transcriptome:
counts vs. semi-quantitative light signal intensities
• Higher dynamic range: log2>6 vs. log2<3
• Rare transcripts are exactly quantified
Digital Gene Expression vs. Microarrays
SuperTAG-DGE includes rare Transcripts
About 80–95% of all mRNA species are present in
five or fewer copies per cell. These rare transcripts
make up 35–50% of all the mRNAs.
SuperSAGE-Analysis: Transcript Frequencies
Example: 3.455.653 Tags from Mouse Spleen (Mus musculus)
101-1000
0,41%
6-20
17%
1000-10.000
0,16%
21-100
8%
>10.000
0,01%
More than 75 % rare transcripts:
Only this part
1
is visible
for
43%
microarrays
This information
is lost on microarrays !
2-5, 32%
>18.000 different transcripts excluding the singletons
* >13.000 Singletons with distinct matches to the NCBI-DB
SuperTAG vs. Micro-arrays
Comparable data:
Exact number for every transcript vs. semiquantitative values
(Microarrays, RT-PCR)
Detection of antisense RNAs
Stress-regulation of expression of peroxidase antisense transcripts in
Cicer arietinum (chickpea)
Expression profiles of peroxidase gene family Antisense (AS) and
Sense (S) transcripts
Expression Ratio
1,5
1
0,5
2-fold
regulation
0
-0,5
-1
-1,5
-2
Drought Stressed Root
-2,5
AS-P1
S-P1
AS-P2
S-P2
SuperTag
AS-P3
S-P3
Salt stressed Root
Salt Stressed Nodule
Normalization of cDNA libraries:
Frequent transcripts are strongly reduced
cDNA before normalization
cDNA after normalization
Analysis of normalized cDNA ends:
Lower costs, sufficient for genotyping!
cDNA before normalisation
Normalized cDNA-Ends:
RNAseq vs. ST-DGE (SuperSAGE)
Mean transcript size : 2 500 bp
AAAAAAA-3’
TTTTTTT-5’
5’cDNA
3’
Tag size:
(
)
26 bp
For the same depth of analysis, about
(50-)100 times more sequencing is required
Functional annotation
Function ?
superTags
nBLAST
nBLAST
cDNA
cDNA Ends
nBLAST
nBLAST
BLASTx
1.
2.
3.
4.
Closest related organism
Lesser related organism
Lesser related organism
Etc.
BLASTx
Swissprot, Trembl,
NCBI
References
Unravelling the interaction of HCMV with dendritic cells using SuperSAGE
M.J. Raftery, E. M. Buchner, H.Matsumura, T.Giese, A. Winkelmann, M. Reuter, R.Terauchi, G.Schönrich and D. H Krüger
J Gen Virol (2009), DOI 10.1099/vir.0.010538-0
Molecular signatures of apomictic and sexual ovules in the Boechera holboellii complex
Timothy F. Sharbel, Marie-Luise Voigt, Jose´ Maria Corral, Thomas Thiel, Alok Varshney, Jochen Kumlehn,
Heiko Vogel and Björn Rotter (2009) The Plant Journal, doi: 10.1111/j.1365-313X.2009.03826.x
Long-Short-Long Games in mRNA Identification: The Length Matters
Wang . S. M. (2008) Current Pharmaceutical Biotechnology, 9, 362-367
SuperSAGE: the drought stress-responsive transcriptome of chickpea roots
Molina C.M., Rotter B., Horres R., Udupa S., Besser B., Bellarmino L., Baum M., Matsumura H., Terauchi R., Kahl G. and Winter P.
(2008) BMC Genomics , 9:553doi:10.1186/1471-2164-9-553
Spermine signaling plays a significant role in the defense response of Arabidopsis thaliana to cucumber mosaic virus.
Mitsuya Y, Takahashi Y, Berberich T, Miyazaki A, Matsumura H, Takahashi H, Terauchi R, Kusano T. (2008)
J Plant Physiol. Oct 13.
SuperSAGE: a modern platform for genome-wide quantitative transcript profiling.
Matsumura H, Krüger DH, Kahl G, Terauchi R.
Curr Pharm Biotechnol. 2008 Oct;9(5):368-74.
SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays. Matsumura H, Bin Nasir KH, Yoshida
K, Ito A, Kahl G, Kruger DH, Terauchi R. (2006) Nat Methods 3:469-474.
Gene expression analysis of plant host-pathogen interactions by SuperSAGE. Matsumura H, Reich S, Ito A, Saitoh H, Kamoun S,
Winter P, Kahl G, Reuter M, Kruger DH, Terauchi R. 2003 Proc Natl Acad Sci U S A. 100:15718-1523.
Thank you for your attention !
Our expertise at your demand
www.genxpro.de