GenXPro

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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

- Degradome

Genomic DNA: - Digital karyotyping (DK), copy number variations (CNVs)- Methylation-specific DK (MSDK)

- Genotyping

- Identification of SNPs

- Molecular markers

Our Service Portfolio

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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 andtranscript quantification.

Transcriptome Analysis & Gene Discovery

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5

3

AAAAAAA-3

TTTTTTT-5

cDNA

cDNA

cDNA

cDNA

Streptavidin-Beads

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3

5

3

5

3

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

Tagging EnzymeAnchoring Enzyme

Sequencing of

Millions of 26

bp SuperTags

Counting, BLAST, Statistics

How it works

SuperTag Digital Gene Expression (STDGE) profiling:

What gene is expressed and how often?

ST-DGE - SuperSAGE became better

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AAAAAAA-3

TTTTTTT-5cDNA

cDNA

cDNA

cDNA

Streptavidin-Beads

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3

5

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5

3

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

1.Digestion with Anchoring Enzyme

What Gene is expressed and how often ?

Digital Gene Expression Profiling

Principle

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AAAAAAA-3

TTTTTTT-5cDNA

cDNA

cDNA

cDNA


Streptavidin-Beads

5

3

5

3

5

3

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

Principle



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Principle

2. First Linker LigationLinker 1

Linker 1

Linker 1

Linker 1

3. Digestion with Tagging Enzyme

4. Recovery of Linker-Tags



AAAAAAA-3

TTTTTTT-5cDNA

cDNA

cDNA

cDNA

Streptavidin-Beads

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

Highly specific 26bp SuperTags

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Principle

2. First Linker LigationLinker 1

Linker 1

Linker 1

Linker 1





AAAAAAA-3

TTTTTTT-5

Streptavidin-Beads

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

5. Second Linker Ligation

Linker 2

Linker 2

Linker 2

Linker 2

5. PCR

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Principle

2. First Linker Ligation





AAAAAAA-3

TTTTTTT-5

Streptavidin-Beads

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

AAAAAAA-3

TTTTTTT-5

6. 2nd-Generation Sequencing

Sequencing of Millions of Tags

7. Counting of Tags, Bioinformatics

Counting, BLAST

5. Second Linker Ligation

5. PCR

Linker 1

Linker 1

Linker 1

Linker 1

Linker 2

Linker 2

Linker 2

Linker 2

Linker 1 Linker 2

Linker 1 Linker 2

Linker 1 Linker 2Linker 1 Linker 2



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Quality of digital gene expression data depends on:

1. Quality of the Tag (what gene is expressed?)

Quality

SuperTag Digital Gene Expression Profiling

2. Quantity of the Tags (how often is the gene expressed?)

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The Tagging Enzyme determines Quality of Tags:

LongSAGE, other DGE platforms

MmeI:5- GGGACNNNNNNNNNNNNNNNNNNNN -3

3- CCCTGNNNNNNNNNNNNNNNNNN -5

5-CAGCAGNNNNNNNNNNNNNNNNNNNNNNNNN -3

3-GTCGTCNNNNNNNNNNNNNNNNNNNNNNNNNNN -5

SuperSAGE, SuperTag-DGE

EcoP15I : 26-27 bp (=SuperTAG)

18-21 bp

Tag-Quality

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What gene?

SuperTags allow unequivocal identification

of the corresponding gene

Tag Quality

Enzyme Plattform Tag-Size e-value

BsmFI-Tag SAGE 14 bp 105

MmeI-Tag LongSAGE, other platforms 18-20 bp 0,34

EcoP15I-Tag SuperSAGE, ST-DGE 26-27 bp 0,00001

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21 bp versus 26 bp

Advantages of the SuperTAG

Only the 26 bp tag can differentiate between the transcripts !

BLAST-Hit , Mus musculus, Score = 52

CATGGTGGCTCACAACCATC Immunoglobulin kappa chain complex

CATGGTGGCTCACAACCATC Tumor necrosis factor (ligand) superfamily, member 10

CATGGTGGCTCACAACCATC Homeodomain leucine zipper-encoding gene

CATGGTGGCTCACAACCATC Mannose phosphate isomerase 1, transcript variant 4

18-20bp (MmeI, LongSAGE)

26 bp (Ecop15I, SuperTAG)

Tag Quality

CATAAC

CGTAAT

TGTAGA

TGTATC

?

?

?

?

!

!

!

!

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Problem of PCR-introduced BIAS

Certain tags are preferentially amplified during PCR

biased quantification

The Solution: GenXProsbias-proof adapters (patent pending)

secure quantification

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26 bp SuperTAGs can:

serve as specific probes: identification of genomic

or cDNA clones

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.

be directly spotted on a microarray for HT analysis1

be used for the simultaneous analysis of two or

more organisms (pathogen/host)2

2. Matsumura et al. (2003) PNAS 100: 15718-15723

1. Matsumura et al. (2006) Nature Methods 3:469-474

Advantages of the SuperTAG

Downstream applications &

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RNA-Seq vs. ST-DGE

(deepSuperSAGE)

For the same depth of analysis, RNA-Seq requires

20-100 times more sequencing !!

Mean transcript size : 2 500 bp

5

3

AAAAAAA-3

TTTTTTT-5cDNA

SuperTag size: 26 bp

*Asmann et. al 2009

STDGE

RNA-Seq

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Normalization of cDNA libraries

Transcript frequencies in human pancreas

Frequencies of transcript species Total transcript distribution

Frequent transcripts make up 50 % of

all transcripts:

To get the info of rare transcripts, these

50% need to be sequenced as well...

Most of the transcript species are

expressed at low levels (below 10 copies

per million).

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Digital Gene Expression vs. Microarrays

Major Advantages of SuperTAG-DGE versus Microarrays

Reliable quantification of the transcriptome:

counts vs. semi-quantitative light signal intensities

Open architecture platform: any gene detected, novel

genes, unexpected transcripts, antisense transcripts

No false positives, no cross hybridisation

Rare transcripts are exactly quantified

Higher dynamic range: unlimited vs. log2

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About 8095% of all mRNA species are present in

five or fewer copies per cell. These rare transcripts

make up 3550% of all the mRNAs.

SuperTAG-DGE includes rare Transcripts

Digital Gene Expression vs. Microarrays

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SuperSAGE-Analysis: Transcript Frequencies

Example: 4.455.653 Tags from Mouse Spleen (Mus muscu lus)*

More than 75 % rare transcripts:

This information

is lost on microarrays !

143%

2-5, 32%

6-2017%

21-1008%

101-10000,41%

1000-10.0000,16% >10.000

0,01%

>18.000 different transcripts excluding the singletons

* >13.000 Singletons with distinct matches to the NCBI-DB

Only this part

is visible for

microarrays

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-7

-6

-4

-3

-2

-1

1

2

3

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Log2 foldchange Taqman assays vs. ST-DGE*

Log2 foldchange SuperSAGE Log2 foldchange Taqman

ST-DGEA Genome-Wide TaqMan Assay

Invariably expressed house

keeping gene Aurora-Kinase A

Similar expression tendency in TaqMan assays and ST-DGE

*in developing chicken embryo gonads

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Comparable data:

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

SuperTAG vs. Micro-arrays

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AAAAAAA-3

AAAAAAA-3

AAAAAAA-3

AAAAAAA-3

microRNA mRNA-ends

Next-Gen-Sequencing, counting, BLAST

microRNAs and the degradome

mRNA

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Digital Karyotyping (DK)

5

3DNA

Quantification of short fragments of genomic DNAto identify chromosomal changes, amplifications, deletions, and the

presence of foreign DNA sequences.

3

5

1.First enzyme digestion

(methylation-sensitive)

5

3

2. First linker ligation, binding to matrix

Biotin

Methylation-specific Digital Karyotyping (MS-DK)

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Methylation-specificDigital Karyotyping (DK)

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3.

Biotin

Second enzyme digestion (methylation-insensitive)

4.

Biotin

Second linker ligation, EcoP15I digestion

SequencingCounting,

AnnotationSuperTag 26bp

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Digital Karyotyping (DK)

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Thank you for your attention

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