1 / 25

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

minor
Télécharger la présentation

Nucleotide - based information

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. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related