Slide 1

hordeprobableΒιοτεχνολογία

4 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

60 εμφανίσεις

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







-

M
olecular

markers


Our

Service Portfolio

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
.

Transcriptome

Analysis & Gene Discovery

5’

3’

AAAAAAA
-
3’

TTTTTTT
-
5’


cDNA


cDNA

cDNA


cDNA

Streptavidin
-
Beads

5’

3’

5’

3’

5’

3’

AAAAAAA
-
3’

TTTTTTT
-
5’

AAAAAAA
-
3’

TTTTTTT
-
5’

AAAAAAA
-
3’

TTTTTTT
-
5’

Tagging Enzyme

Anchoring 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


5’

3’

AAAAAAA
-
3’

TTTTTTT
-
5’

cDNA

cDNA

cDNA

cDNA

Streptavidin
-
Beads

5’

3’

5’

3’

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

5’

3’

AAAAAAA
-
3’

TTTTTTT
-
5’

cDNA

cDNA

cDNA

cDNA

Digital Gene Expression Profiling

Streptavidin
-
Beads

5’

3’

5’

3’

5’

3’

AAAAAAA
-
3’

TTTTTTT
-
5’

AAAAAAA
-
3’

TTTTTTT
-
5’

AAAAAAA
-
3’

TTTTTTT
-
5’

Principle

1.Digestion with Anchoring Enzyme

What Gene is expressed and how often ?

Digital Gene Expression Profiling

Principle

2. First Linker Ligation

Linker 1

Linker 1

Linker 1

Linker 1

3. Digestion with Tagging Enzyme

4. Recovery of Linker
-
Tags

What Gene is expressed and how often ?

1.Digestion with Anchoring Enzyme

AAAAAAA
-
3’

TTTTTTT
-
5’

cDNA

cDNA

cDNA

cDNA

Streptavidin
-
Beads

AAAAAAA
-
3’

TTTTTTT
-
5’

AAAAAAA
-
3’

TTTTTTT
-
5’

AAAAAAA
-
3’

TTTTTTT
-
5’

Highly specific 26bp “SuperTags“

Digital Gene Expression Profiling

Principle

2. First Linker Ligation

Linker 1

Linker 1

Linker 1

Linker 1

3. Digestion with Tagging Enzyme

4. Recovery of Linker
-
Tags

What Gene is expressed and how often ?

1.Digestion with Anchoring Enzyme

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

Digital Gene Expression Profiling

Principle

2. First Linker Ligation

3. Digestion with Tagging Enzyme

4. Recovery of Linker
-
Tags

What Gene is expressed and how often ?

1.Digestion with Anchoring Enzyme

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 2

Linker 1

Linker 2

Linker 1

Linker 2

Linker 1

Linker 2

Linker 1

Linker 2

Linker 1

Linker 2

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
?)

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

Eco
P
15
I :



26
-
27
bp

(=
SuperTAG
)


18
-
21
bp

Tag
-
Quality

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

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

C
A
T
GG
T
GG
C
T
C
A
C
AA
CC
A
T
C


Immunoglobulin

kappa
chain

complex

C
A
T
GG
T
GG
C
T
C
A
C
AA
CC
A
T
C


Tumor necrosis factor (
ligand
)
superfamily
, member 10

C
A
T
GG
T
GG
C
T
C
A
C
AA
CC
A
T
C


Homeodomain

leucine

zipper
-
encoding

gene


C
A
T
GG
T
GG
C
T
C
A
C
AA
CC
A
T
C


Mannose

phosphate

isomerase

1,
transcript

variant
4


18
-
20bp (MmeI, LongSAGE)


26 bp (Ecop15I, SuperTAG)

Tag Quality

C
A
T
AA
C

C
G
T
AA
T

T
G
T
A
G
A

T
G
T
A
T
C

?

?

?

?

!

!

!

!

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

26
bp

SuperTAGs

can:


s
erve
as specific probes: identification of genomic
or
cDNA

clones


d
irectly
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 analysis
1


b
e
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 &

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
-
5’

cDNA

SuperTag

size:




26
bp


*
Asmann

et. al 2009

STDGE

RNA
-
Seq

Normalization of
cDNA

libraries

Transcript frequencies in human pancreas

Frequencies

of

transcript

species

Total
t
ranscript

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
).

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. log
2
<3



Simultaneous analysis of more than one organisms: parasite
-
host


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.


SuperTAG
-
DGE includes
rare Transcripts

Digital Gene Expression vs. Microarrays

SuperSAGE
-
Analysis:
Transcript

Frequencies

Example
:
4.455.653
Tags
from

Mouse Spleen (
Mus
musculus
)*





More than 75 % rare transcripts:

This information

is
lost on microarrays !

1

43%

2
-
5, 32%

6
-
20

17%

21
-
100

8%

101
-
1000


0,41%

1000
-
10.000


0,16%

>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

-8
-7
-6
-4
-3
-2
-1
1
2
3
5
Log2
foldchange

Taqman

assays vs
.
ST
-
DGE*

Log2 foldchange SuperSAGE
Log2 foldchange Taqman
ST
-
DGE

A 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

Comparable data:

Exact number for every transcript vs.
semiquantitative

values
(Microarrays, RT
-
PCR)


SuperTAG

vs. Micro
-
arrays

AAAAAAA
-
3


AAAAAAA
-
3


AAAAAAA
-
3


AAAAAAA
-
3


microRNA

mRNA
-
ends

Next
-
Gen
-
Sequencing
,
counting
, BLAST

microRNAs

and the
degradome

mRNA

Digital Karyotyping (DK)

5‘

3‘

DNA

Quantification
of short fragments of genomic DNA

to 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
l
igation
,
binding

to

matrix

Biotin

Methylation
-
specific Digital
Karyotyping

(MS
-
DK
)

Methylation
-
specificDigital

Karyotyping

(DK)

5


3


3.

Biotin

Second
enzyme

digestion

(
methylation
-
insensitive
)

4.

Biotin

Second linker
ligation
,
EcoP15I
digestion

Sequencing

Counting,

Annotation

SuperTag 26bp

5‘

3‘

Digital Karyotyping (DK)



Thank you for your
attention

www.genxpro.de