[email protected] GenXPro GmbH, Frankfurt am Main Our expertise at your demand.

[email protected]

GenXPro GmbH, Frankfurt am Main

www.genxpro.de

Our expertise at your demand

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.

Thank you for your attention !

Our expertise at your demand

www.genxpro.de