slide1 l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
info@genxpro.de PowerPoint Presentation
Download Presentation
info@genxpro.de

Loading in 2 Seconds...

play fullscreen
1 / 27

info@genxpro.de - PowerPoint PPT Presentation


  • 81 Views
  • Uploaded on

Our expertise at your demand. info@genxpro.de. 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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'info@genxpro.de' - page


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

Our expertise at your demand

info@genxpro.de

GenXPro GmbH, Frankfurt am Main

www.genxpro.de

slide2

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
slide3

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.

slide4

Digital Gene expression Profiling

Principle

What Gene is expressed and how often ?

Anchoring Enzyme

Streptavidin-Beads

Tagging Enzyme

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

Sequencing of Millions of 26 bp SuperTags

Counting, BLAST

slide5

Digital Gene expression Profiling

Principle

Streptavidin-Beads

1.Digestion with Anchoring Enzyme

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

slide6

Digital Gene Expression Profiling

Principle

What Gene is expressed and how often ?

Streptavidin-Beads

1.Digestion with Anchoring Enzyme

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

slide7

Digital Gene Expression Profiling

Principle

What Gene is expressed and how often ?

Streptavidin-Beads

1.Digestion with Anchoring Enzyme

AAAAAAA-3’

TTTTTTT-5’

Linker 1

cDNA

2. First Linker Ligation

3. Digestion with Tagging Enzyme

AAAAAAA-3’

TTTTTTT-5’

Linker 1

cDNA

4. Recovery of Linker-Tags

AAAAAAA-3’

TTTTTTT-5’

Linker 1

cDNA

AAAAAAA-3’

TTTTTTT-5’

Linker 1

cDNA

Highly specific 26bp “SuperTags“

slide8

Digital Gene Expression Profiling

Principle

What Gene is expressed and how often ?

Streptavidin-Beads

1.Digestion with Anchoring Enzyme

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

2. First Linker Ligation

3. Digestion with Tagging Enzyme

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

4. Recovery of Linker-Tags

5. Second Linker Ligation

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

5. PCR

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

slide9

Digital Gene Expression Profiling

Principle

What Gene is expressed and how often ?

Streptavidin-Beads

1.Digestion with Anchoring Enzyme

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

2. First Linker Ligation

Linker 1

Linker 2

Linker 1

Linker 2

3. Digestion with Tagging Enzyme

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

Linker 1

Linker 2

4. Recovery of Linker-Tags

Linker 1

Linker 2

Sequencing of Millions of Tags

5. Second Linker Ligation

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

Linker 1

Linker 2

5. PCR

Linker 1

Linker 2

6. Next-Generation Sequencing

AAAAAAA-3’

TTTTTTT-5’

Linker 1

Linker 2

Linker 1

Linker 2

7. Counting of Tags, Bioinformatics

Linker 1

Linker 2

Counting, BLAST

slide10

Digital Gene expression Profiling

Principle

Anchoring Enzyme

Streptavidin-Beads

Tagging Enzyme

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

5’

3’

AAAAAAA-3’

TTTTTTT-5’

cDNA

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

Sequencing of Millions of 26 bpSuperTags

Counting, BLAST

quality of digital gene expression data depends on

Digital Gene Expression Profiling

Quality

Quality of digital gene expression data depends on:

1. Quality ofthe Tag (whatgeneisexpressed?)

2. Quantityofthe Tags (howoftenisthegeneexpressed?)

slide12

Tag-Quality

The Tagging Enzyme determines Quality of Tags:

LongSAGE, other DGE platforms

MmeI:

18-20 bp

5’- GGGACNNNNNNNNNNNNNNNNNNNN -3’

3’- CCCTGNNNNNNNNNNNNNNNNNN -5’

SuperSAGE, SuperTAG-DGE

EcoP15I : 26 bp (=SuperTAG)‏

5’-CAGCAGNNNNNNNNNNNNNNNNNNNNNNNN -3’

3’-GTCGTCNNNNNNNNNNNNNNNNNNNNNNNNNN -5’

slide13

Tag Quality

What gene?

SuperTAGs allow unequivocal Identification

of the corresponding Gene

slide14

Tag Quality

Advantages of the SuperTAG

20 bp versus 26 bp

18-20bp (MmeI, LongSAGE)

26 bp (Ecop15I, SuperTAG)

Only the 26 bp tag can differentiate between the transcripts !

slide15

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

slide16

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

Matsumura et al. (2006) Nature Methods 3:469-474

2. Matsumura et al. (2003) PNAS 100: 15718-15723

slide17

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
slide18

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.

slide19

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 !

Only this part is visible for microarrays

>18.000 different transcripts excluding the singletons

* >13.000 Singletons with distinct matches to the NCBI-DB

slide20

SuperTAG vs. Micro-arrays

Comparable data:

Exact number for every transcript vs. semiquantitative values (Microarrays, RT-PCR)‏

slide21

Detection of antisense RNAs

Stress-regulation of expression of peroxidase antisense transcripts in

Cicer arietinum (chickpea)

2-fold

regulation

slide22

Normalization of cDNA libraries:

Frequent transcripts are strongly reduced

cDNA before normalization

cDNA after normalization

slide23

Analysis of normalized cDNA ends:

Lower costs, sufficient for genotyping!

cDNA before normalisation

Normalized cDNA-Ends:

slide24

RNAseq vs. ST-DGE (SuperSAGE)

Mean transcript size : 2 500 bp

Tag size: ( ) 26 bp

AAAAAAA-3’

TTTTTTT-5’

5’

3’

cDNA

For the same depth of analysis, about

(50-)100 times more sequencing is required

slide25

Functional annotation

Function ?

superTags

cDNA

cDNA Ends

nBLAST

nBLAST

nBLAST

nBLAST

BLASTx

BLASTx

  • Closest related organism
  • Lesser related organism
  • Lesser related organism
  • Etc.

Swissprot, Trembl, NCBI

slide26

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 Boecheraholboellii complex

Timothy F. Sharbel, Marie-Luise Voigt, Jose´ Maria Corral, Thomas Thiel, AlokVarshney, 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: thedrought stress-responsivetranscriptomeofchickpearoots

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

Sperminesignalingplays a significantrole in thedefenseresponseofArabidopsisthalianatocucumbermosaicvirus.

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.

CurrPharmBiotechnol. 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 NatlAcadSci U S A. 100:15718-1523.

slide27

Thank you for your attention !

Our expertise at your demand

www.genxpro.de