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info@genxpro.de

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

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info@genxpro.de

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  1. Our expertise at your demand info@genxpro.de GenXPro GmbH, Frankfurt am Main www.genxpro.de

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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“

  8. 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

  9. 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

  10. 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

  11. Digital Gene Expression Profiling Quality Quality of digital gene expression data depends on: 1. Quality ofthe Tag (whatgeneisexpressed?) 2. Quantityofthe Tags (howoftenisthegeneexpressed?)

  12. 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’

  13. Tag Quality What gene? SuperTAGs allow unequivocal Identification of the corresponding Gene

  14. 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 !

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. SuperTAG vs. Micro-arrays Comparable data: Exact number for every transcript vs. semiquantitative values (Microarrays, RT-PCR)‏

  21. Detection of antisense RNAs Stress-regulation of expression of peroxidase antisense transcripts in Cicer arietinum (chickpea) 2-fold regulation

  22. Normalization of cDNA libraries: Frequent transcripts are strongly reduced cDNA before normalization cDNA after normalization

  23. Analysis of normalized cDNA ends: Lower costs, sufficient for genotyping! cDNA before normalisation Normalized cDNA-Ends:

  24. 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

  25. 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

  26. 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.

  27. Thank you for your attention ! Our expertise at your demand www.genxpro.de

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