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info@genxpro.de

info@genxpro.de. GenXPro GmbH, Frankfurt am Main www.genxpro.de. Nucleotide based information:. mRNA SuperSAGE qRT -PCR, Taq -Man assays, Real-Time PCR service - 3‘- and 5‘- RACE - Normalization of cDNA libraries ( qual . Information) 2) non coding RNA ( microRNA )

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info@genxpro.de

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  1. info@genxpro.de GenXPro GmbH, Frankfurt am Main www.genxpro.de

  2. Nucleotide based information: • mRNA • SuperSAGE • qRT-PCR, Taq-Man assays, Real-Time PCR service • - 3‘- and 5‘- RACE • - NormalizationofcDNAlibraries (qual. Information) • 2) non coding RNA (microRNA) • Genomic DNA: - Digital karyotyping (DK) • - MethylationspecificDK (MSDK) • - Genotyping • - Identificationof SNPs • - molecularmarkers Transcription :

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  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 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“

  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 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

  10. 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

  11. 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

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

  13. 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’

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

  15. 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 !

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  21. 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

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

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

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

  25. Normalization of cDNA libraries Transcript frequencies Frequencies of transcript species Total transcript distribution Frequent transcripts make up 50 % of all transcripts. Most of the transcript species are expressed at low levels (below 10 copies per million). Normalization is useful for qualitative whole transcriptome sequencing

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

  27. microRNAs and the degradome microRNA mRNA-ends AAAAAAA-3’ AAAAAAA-3’ mRNA AAAAAAA-3’ AAAAAAA-3’ Next-Gen-Sequencing, counting, BLAST

  28. microRNAs and the degradome microRNA-sequencing Sequencing of Millions of microRNAs Counting, BLAST, analysis of differential expression PARE: parallel analysis of RNA ends Sequencing of Millions of uncapped 5‘ ends Counting, BLAST, analysis of differential expression

  29. 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

  30. Digital Karyotyping (DK) Quantification of short fragments of genomic DNA to identify chromosomal changes, amplifications, deletions, and the presence of foreign DNA sequences. First enzyme digestion (methylation insensitive) 1. 3‘ 5‘ DNA 5‘ 3‘ 2. First Linker Ligation, binding to matrix 5‘ 3‘ Biotin

  31. Digital Karyotyping (DK) 3. Second enzyme digestion (methylation sensitive) 5‘ 3‘ Biotin 4. Second linker ligation, Ecop15I digestion 5‘ 3‘ Biotin Counting, Annotation SuperTag 26bp Sequencing

  32. Methylation specific Digital Karyotyping (MSDK) Genome-wide DNA methylation analysis Methylation sensitive enzyme 1. 3‘ 5‘ DNA 5‘ 3‘ 2. First Linker Ligation, binding to matrix 5‘ 3‘ Biotin

  33. Methylation specific Digital Karyotyping (MSDK) 3. Second enzyme digestion 5‘ 3‘ Biotin 4. Second linker ligation, Ecop15I digestion 5‘ 3‘ Biotin Counting, Annotation SuperTag 26bp Sequencing Ditagformation

  34. 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.

  35. Thank you for your attention ! www.genxpro.de

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