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TRANSCRIPT
Our Service Portfolio
- Digital Gene Expression Service: from cells/tissues to annotated/BLASTed libraries inone 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
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.
Transcriptome Analysis & Gene Discovery
5’3’
AAAAAAA-3’TTTTTTT-5’cDNA
cDNA
cDNA
cDNA
Streptavidin-Beads
5’3’
5’3’
5’3’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
Tagging EnzymeAnchoring Enzyme
Sequencing of Millions of 26 bp SuperTags
Counting, BLAST
Digital Gene expression ProfilingPrinciple
What Gene is expressed and how often ?
5’3’
AAAAAAA-3’TTTTTTT-5’cDNA
cDNA
cDNA
cDNA
Streptavidin-Beads
5’3’
5’3’
5’3’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
1.Digestion with Anchoring Enzyme
Digital Gene expression ProfilingPrinciple
5’3’
AAAAAAA-3’TTTTTTT-5’cDNA
cDNA
cDNA
cDNA
Digital Gene Expression Profiling
Streptavidin-Beads
5’3’
5’3’
5’3’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
Principle
1.Digestion with Anchoring Enzyme
What Gene is expressed and how often ?
Digital Gene Expression ProfilingPrinciple
2. First Linker Ligation Linker 1
Linker 1
Linker 1
Linker 1
3. Digestion with Tagging Enzyme
4. Recovery of Linker-Tags
What Gene is expressed and how often ?
1.Digestion with Anchoring EnzymeAAAAAAA-3’TTTTTTT-5’cDNA
cDNA
cDNA
cDNA
Streptavidin-Beads
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
Highly specific 26bp “SuperTags“
Digital Gene Expression ProfilingPrinciple
2. First Linker Ligation Linker 1
Linker 1
Linker 1
Linker 1
3. Digestion with Tagging Enzyme
4. Recovery of Linker-Tags
What Gene is expressed and how often ?
1.Digestion with Anchoring EnzymeAAAAAAA-3’TTTTTTT-5’
Streptavidin-Beads
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
5. Second Linker Ligation
Linker 2
Linker 2
Linker 2
Linker 25. PCR
Digital Gene Expression ProfilingPrinciple
2. First Linker Ligation
3. Digestion with Tagging Enzyme
4. Recovery of Linker-Tags
What Gene is expressed and how often ?
1.Digestion with Anchoring EnzymeAAAAAAA-3’TTTTTTT-5’
Streptavidin-Beads
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
6. Next-Generation Sequencing
Sequencing of Millions of Tags
7. Counting of Tags, Bioinformatics
Counting, BLAST
5. Second Linker Ligation
5. PCR
Linker 1
Linker 1
Linker 1
Linker 1
Linker 2
Linker 2
Linker 2
Linker 2
Linker 1 Linker 2Linker 1 Linker 2
Linker 1 Linker 2Linker 1 Linker 2
Linker 1 Linker 2Linker 1 Linker 2
Linker 1 Linker 2 Linker 1 Linker 2
5’3’
AAAAAAA-3’TTTTTTT-5’cDNA
cDNA
cDNA
cDNA
Streptavidin-Beads
5’3’
5’3’
5’3’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
AAAAAAA-3’TTTTTTT-5’
Tagging EnzymeAnchoring Enzyme
Sequencing of Millions of 26 bp SuperTags
Sequencing of Millions of 26 bp SuperTags
Counting, BLASTCounting, BLAST
Digital Gene expression ProfilingPrinciple
Quality of digital gene expression data depends on:
1. Quality of the Tag (what gene is expressed?)
QualityDigital Gene Expression Profiling
2. Quantity of the Tags (how often is the gene expressed?)
The Tagging Enzyme determines Quality of Tags:
LongSAGE, other DGE platforms
MmeI:
5’- GGGACNNNNNNNNNNNNNNNNNNNN -3’ 3’- CCCTGNNNNNNNNNNNNNNNNNN -5’
5’-CAGCAGNNNNNNNNNNNNNNNNNNNNNNNN -3’ 3’-GTCGTCNNNNNNNNNNNNNNNNNNNNNNNNNN -5’
SuperSAGE, SuperTAG-DGE
EcoP15I : 26 bp (=SuperTAG)
18-20 bp
Tag-Quality
What gene?
SuperTAGs allow unequivocal Identification
of the corresponding Gene
Tag Quality
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
20 bp versus 26 bp
Advantages of the SuperTAG
Only the 26 bp tag can differentiate between the transcripts !
BLAST-Hit , Mus musculus, Score = 52
CATGGTGGCTCACAACCATC Immunoglobulin kappa chain complex
CATGGTGGCTCACAACCATC Tumor necrosis factor (ligand) superfamily, member 10
CATGGTGGCTCACAACCATC Homeodomain leucine zipper-encoding gene
CATGGTGGCTCACAACCATC Mannose phosphate isomerase 1, transcript variant 4
18-20bp (MmeI, LongSAGE)
26 bp (Ecop15I, SuperTAG)
Tag Quality
CATAAC
CGTAAT
TGTAGA
TGTATC
?
?
?
?
!
!
!
!
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
26 bp SuperTAGs can:
• Serve as specific probes: identification of genomic or cDNA clones
• 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.
• Be directly spotted on a microarray for HT analysis1
•Be used for the simultaneous analysis of two or more organisms (pathogen/host)2
2. Matsumura et al. (2003) PNAS 100: 15718-15723
1. Matsumura et al. (2006) Nature Methods 3:469-474
Advantages of the SuperTAG
Downstream applications &
Digital Gene Expression vs. Microarrays
Major Advantages of SuperTAG-DGE versus Microarrays
• Reliable quantification of the transcriptome:
counts vs. semi-quantitative light signal intensities
• Open architecture platform: any gene detected, novel
genes, unexpected transcripts, antisense transcripts
• No false positives, no cross hybridisation
• Rare transcripts are exactly quantified
• Higher dynamic range: log2>6 vs. log2<3
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.
SuperTAG-DGE includes rare Transcripts
Digital Gene Expression vs. Microarrays
SuperSAGE-Analysis: Transcript Frequencies
Example: 3.455.653 Tags from Mouse Spleen (Mus musculus)
More than 75 % rare transcripts:
This information
is lost on microarrays !
>18.000 different transcripts excluding the singletons
* >13.000 Singletons with distinct matches to the NCBI-DB
Only this part is visible for microarrays
Comparable data:
Exact number for every transcript vs. semiquantitative values (Microarrays, RT-PCR)
SuperTAG vs. Micro-arrays
-2,5
-2
-1,5
-1
-0,5
0
0,5
1
1,5
AS-P1 S-P1 AS-P2 S-P2 AS-P3 S-P3
Expression profiles of peroxidase gene family Antisense (AS) and Sense (S) transcripts
Drought Stressed Root
Salt stressed Root
Salt Stressed NoduleSuperTag
Exp
ress
ion
Rat
io
2-fold regulation
Detection of antisense RNAs
Stress-regulation of expression of peroxidase antisense transcripts in Cicer arietinum (chickpea)
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)
For the same depth of analysis, about (50-)100 times more sequencing is required
Mean transcript size : 2 500 bp
Tag size: ( ) 26 bp
5’3’
AAAAAAA-3’TTTTTTT-5’cDNA
Functional annotation
cDNA EndssuperTags cDNA
Function ?
Swissprot, Trembl, NCBI
nBLAST nBLAST
nBLAST
BLASTxnBLAST
1. Closest related organism2. Lesser related organism3. Lesser related organism4. Etc.
BLASTx
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 complexTimothy 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 MattersWang . S. M. (2008) Current Pharmaceutical Biotechnology, 9, 362-367
SuperSAGE: the drought stress-responsive transcriptome of chickpea rootsMolina 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.
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