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 - Degradome

Genomic DNA: - Digital karyotyping (DK), copy number variations (CNVs) - Methylation-specific DK (MSDK) - Genotyping - Identification of SNPs - Molecular markers

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.

ST-DGE - SuperSAGE became better How it works

SuperTag Digital Gene Expression (STDGE) profiling: What gene is expressed and how often?
Anchoring Enzyme
5 3 5 3 5 3 5 3

Tagging Enzyme
cDNA

Streptavidin-Beads AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5

cDNA cDNA cDNA

Sequencing of Millions of 26 bp SuperTags Counting, BLAST, Statistics

Digital Gene Expression Profiling Principle

What Gene is expressed and how often ?
1.Digestion with Anchoring Enzyme 5 3 5 3 5 3 5 3 cDNA
Streptavidin-Beads

AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5

cDNA cDNA

cDNA

Digital Gene Expression Profiling Principle

What Gene is expressed and how often ?
1.Digestion with Anchoring Enzyme 5 3 5 3 5 3 5 3 cDNA
Streptavidin-Beads

AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5

cDNA cDNA

cDNA

Digital Gene Expression Profiling Principle

What Gene is expressed and how often ?
1.Digestion with Anchoring Enzyme 2. First Linker Ligation 3. Digestion with Tagging Enzyme 4. Recovery of Linker-Tags Linker 1 Linker 1 Linker 1 cDNA Linker 1 cDNA
Streptavidin-Beads

AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5

cDNA cDNA

Highly specific 26bp SuperTags

Digital Gene Expression Profiling Principle

What Gene is expressed and how often ?
1.Digestion with Anchoring Enzyme 2. First Linker Ligation 3. Digestion with Tagging Enzyme 4. Recovery of Linker-Tags 5. Second Linker Ligation 5. PCR Linker 1 Linker 2 Linker 1 Linker 1 Linker 2 Linker 2 Linker 1 Linker 2
Streptavidin-Beads

AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5 AAAAAAA-3 TTTTTTT-5

Digital Gene Expression Profiling Principle

What Gene is expressed and how often ?
1.Digestion with Anchoring Enzyme 2. First Linker Ligation 3. Digestion with Tagging Enzyme 4. Recovery of Linker-Tags 5. Second Linker Ligation 5. PCR 6. 2nd-Generation Sequencing 7. Counting of Tags, Bioinformatics Linker 11 Linker Linker 1 Linker 22 Linker Linker 2 Linker 1 Linker 1 Linker 1
Streptavidin-Beads

AAAAAAA-3 Linker 2 TTTTTTT-5 Linker 2 Linker 2 AAAAAAA-3 TTTTTTT-5

Linker 1 Linker 2 Linker 1 Linker 2 Linker 1 Linker 2 Linker 1 Linker 1 Linker 1

Sequencing of Millions of Tags

AAAAAAA-3 TTTTTTT-5

AAAAAAA-3 Linker 2 TTTTTTT-5 Linker 2 Linker 2

Counting, BLAST

SuperTag Digital Gene Expression Profiling Quality

Quality of digital gene expression data depends on:

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

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

Tag-Quality

The Tagging Enzyme determines Quality of Tags:

LongSAGE, other DGE platforms 18-21 bp MmeI: 5- GGGACNNNNNNNNNNNNNNNNNNNN -3 3- CCCTGNNNNNNNNNNNNNNNNNN -5 SuperSAGE, SuperTag-DGE EcoP15I : 26-27 bp (=SuperTAG) -3 -5

5-CAGCAGNNNNNNNNNNNNNNNNNNNNNNNNN 3-GTCGTCNNNNNNNNNNNNNNNNNNNNNNNNNNN

Tag Quality

What gene?
SuperTags allow unequivocal identification of the corresponding gene
Enzyme
BsmFI-Tag MmeI-Tag

Plattform
SAGE LongSAGE, other platforms

Tag-Size
14 bp 18-20 bp

e-value
105 0,34

EcoP15I-Tag

SuperSAGE, ST-DGE

26-27 bp

0,00001

Tag Quality Advantages of the SuperTAG

21 bp versus 26 bp
18-20bp (MmeI, LongSAGE) 26 bp (Ecop15I, SuperTAG) CATGGTGGCTCACAACCATC CATAAC CATGGTGGCTCACAACCATC CGTAAT CATGGTGGCTCACAACCATC TGTAGA CATGGTGGCTCACAACCATC TGTATC
BLAST-Hit , Mus musculus, Score = 52 Immunoglobulin kappa chain complex

? !

! Tumor necrosis factor (ligand) superfamily, member 10 ?

Homeodomain leucine zipper-encoding gene Mannose phosphate isomerase 1, transcript variant 4

? ! ? !

Only the 26 bp tag can differentiate between the transcripts !

Problem of PCR-introduced BIAS

Certain tags are preferentially amplified during PCR

biased quantification

The Solution: GenXPros bias-proof adapters (patent pending)

secure quantification

Downstream applications & Advantages of the SuperTAG 26 bp SuperTAGs 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
1. Matsumura et al. (2006) Nature Methods 3:469-474 2. Matsumura et al. (2003) PNAS 100: 15718-15723

RNA-Seq vs. ST-DGE (deepSuperSAGE)

Mean transcript size : 2 500 bp

5cDNA 3 AAAAAAA-3 TTTTTTT-5

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

Normalization of cDNA libraries Transcript frequencies in human pancreas

Frequencies of transcript species

Total transcript distribution

Most of the transcript species are expressed at low levels (below 10 copies per million).

Frequent transcripts make up 50 % of all transcripts: To get the info of rare transcripts, these 50% need to be sequenced as well...

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

Digital Gene Expression vs. Microarrays

SuperTAG-DGE includes rare Transcripts

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.

SuperSAGE-Analysis: Transcript Frequencies

Example: 4.455.653 Tags from Mouse Spleen (Mus musculus)*

101-1000 0,41%

1000-10.000 0,16% 21-100 8%

>10.000 0,01%

More than 75 % rare transcripts: This information is lost on microarrays !

6-20 17%

Only this part 1 is visible for 43% microarrays

2-5, 32%

>18.000 different transcripts excluding the singletons * >13.000 Singletons with distinct matches to the NCBI-DB

ST-DGE
A Genome-Wide TaqMan Assay
Similar expression tendency in TaqMan assays and ST-DGE Log2 foldchange Taqman assays vs. ST-DGE*
Log2 foldchange SuperSAGE Log2 foldchange Taqman

5 3 2 1 -1 -2 -3 -4 -6 -7 -8

Invariably expressed house keeping gene Aurora-Kinase A

*in developing chicken embryo gonads

SuperTAG vs. Micro-arrays

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

microRNAs and the degradome

microRNA

mRNA-ends
AAAAAAA-3

AAAAAAA-3

mRNA
AAAAAAA-3 AAAAAAA-3

Next-Gen-Sequencing, counting, BLAST

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.
1.
5 3 First enzyme digestion (methylation-sensitive) DNA 3 5

2.
5 3

First linker ligation, binding to matrix

Biotin

Digital Karyotyping (DK) Methylation-specificDigital Karyotyping (DK)

3.
5 3

Second enzyme digestion (methylation-insensitive)

Biotin

4.

Second linker ligation, EcoP15I digestion

5 3

Biotin

SuperTag 26bp

Sequencing

Counting, Annotation

Thank you for your attention

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