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Nucleotide - based information

Our Service Portfolio. Nucleotide - based information. Transcripts : mRNA - SuperSAGE , ST-DGE - RNAseq - qRT -PCR , Taq -Man assays, Real-Time PCR service - Normalization of cDNA libraries ( qualitative information ) non coding RNA - MicroRNA

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Nucleotide - based information

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  1. Our Service Portfolio Nucleotide - basedinformation Transcripts : mRNA - SuperSAGE, ST-DGE - RNAseq - qRT-PCR, Taq-Man assays, Real-Time PCR service - NormalizationofcDNAlibraries (qualitative information) non coding RNA - MicroRNA - Degradome Genomic DNA: - Digital karyotyping (DK), copynumbervariations (CNVs) - Methylation-specific DK (MSDK) - Genotyping - Identificationof SNPs - Molecularmarkers

  2. Transcriptome Analysis & Gene Discovery SuperTag Digital Gene Expression Profiling (ST-DGE) A patented, improved version of SuperSAGE, applying deep sequencing and a bias-free PCR technology for optimal tag-to-gene annotation and transcript quantification.

  3. ST-DGE - SuperSAGEbecamebetter How it works SuperTag Digital Gene Expression (STDGE) profiling: 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 bpSuperTags Counting, BLAST, Statistics

  4. 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

  5. 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

  6. 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“

  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 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

  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 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. 2nd-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

  9. SuperTag Digital Gene Expression Profiling Quality Quality of digital geneexpressiondatadepends on: 1. Quality ofthe Tag (whatgeneisexpressed?) 2. Quantityofthe Tags (howoftenisthegeneexpressed?)

  10. Tag-Quality The Tagging Enzyme determines Quality of Tags: LongSAGE, other DGE platforms MmeI: 18-21 bp 5’- GGGACNNNNNNNNNNNNNNNNNNNN -3’ 3’- CCCTGNNNNNNNNNNNNNNNNNN -5’ SuperSAGE, SuperTag-DGE EcoP15I : 26-27 bp (=SuperTAG)‏ 5’-CAGCAGNNNNNNNNNNNNNNNNNNNNNNNNN -3’ 3’-GTCGTCNNNNNNNNNNNNNNNNNNNNNNNNNNN -5’

  11. Tag Quality What gene? SuperTagsallow unequivocal identification of the corresponding gene

  12. Tag Quality Advantages of the SuperTAG 21 bpversus 26 bp 18-20bp (MmeI, LongSAGE) 26 bp (Ecop15I, SuperTAG) Only the 26 bp tag can differentiate between the transcripts !

  13. 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

  14. Downstream applications & Advantages of the SuperTAG 26 bpSuperTAGs can: • directly be used as highly specific primer for PCR 3‘- and 5‘- RACE,RCA, 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

  15. RNA-Seqvs. ST-DGE (deepSuperSAGE) Mean transcript size : 2 500 bp AAAAAAA-3’ TTTTTTT-5’ 5’ 3’ cDNA RNA-Seq SuperTag size: 26 bp STDGE For the same depth of analysis, RNA-Seq requires 20-100 times more sequencing !! *Asmann et. al 2009

  16. Normalization of cDNAlibraries Transcript frequencies in human pancreas Frequenciesoftranscriptspecies Total transcriptdistribution Frequenttranscriptsmakeup 50 % of all transcripts: Togettheinfoof rare transcripts, these 50% needtobesequencedas well... Most ofthetranscriptspeciesareexpressedatlowlevels (below 10 copies per million).

  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: unlimited vs. log2<3 • Rare transcripts are exactly quantified • Simultaneous analysis of more than one organisms: parasite-host

  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: TranscriptFrequencies Example: 4.455.653 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. ST-DGE A Genome-Wide TaqMan Assay Similarexpressiontendency in TaqMan assays andST-DGE Invariablyexpressedhousekeepinggene Aurora-Kinase A *in developingchickenembryogonads

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

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

  23. Digital Karyotyping (DK) Methylation-specific Digital Karyotyping(MS-DK) Quantification of short fragments of genomic DNA to identify chromosomal changes, amplifications, deletions, and the presence of foreign DNA sequences. First enzymedigestion (methylation-sensitive) 1. 5‘ 3‘ 3‘ 5‘ DNA 2. First linker ligation, bindingtomatrix 5‘ 3‘ Biotin

  24. Digital Karyotyping (DK) Methylation-specificDigitalKaryotyping (DK) 3. Second enzymedigestion (methylation-insensitive) 5‘ 3‘ Biotin 4. Second linker ligation, EcoP15I digestion 5‘ 3‘ Biotin Counting, Annotation SuperTag 26bp Sequencing

  25. Thank you for your attention www.genxpro.de

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