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[email protected] 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

[email protected]

GenXPro GmbH, Frankfurt am Main

www.genxpro.de


Info genxpro de

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 :


    Info genxpro de

    Our Service Portfolio

    • Digital Gene Expression Service:

    • from cells/tissues to annotated/BLASTed libraries inone 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


    Info genxpro de

    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.


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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“


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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?)


    Info genxpro de

    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’


    Info genxpro de

    Tag Quality

    What gene?

    SuperTAGs allow unequivocal Identification

    of the corresponding Gene


    Info genxpro de

    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 !


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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.


    Info genxpro de

    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


    Info genxpro de

    SuperTAG vs. Micro-arrays

    Comparable data:

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


    Info genxpro de

    Detection of antisense RNAs

    Stress-regulation of expression of peroxidase antisense transcripts in

    Cicer arietinum (chickpea)

    2-fold

    regulation


    Info genxpro de

    Normalization of cDNA libraries:

    Frequent transcripts are strongly reduced

    cDNA before normalization

    cDNA after normalization


    Info genxpro de

    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


    Info genxpro de

    Analysis of normalized cDNA ends:

    Lower costs, sufficient for genotyping!

    cDNA before normalisation

    Normalized cDNA-Ends:


    Info genxpro de

    microRNAs and the degradome

    microRNA

    mRNA-ends

    AAAAAAA-3’

    AAAAAAA-3’

    mRNA

    AAAAAAA-3’

    AAAAAAA-3’

    Next-Gen-Sequencing, counting, BLAST


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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


    Info genxpro de

    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.


    Info genxpro de

    Thank you for your attention !

    www.genxpro.de


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