ChronicDisease - uni-muenster.decampus.uni-muenster.de/fileadmin/einrichtung/ifg/IFG_Broschuere.pdf · This information folder provides an overview of the different facilities, their

Technology PlatformInterdisciplinary Center

for Clinical Research

of the Medical Faculty Münster

DiseaseChronic

State-of-the-Art Technologies

IZKF Technology Platform

Our Goal

The rapid advancement in the field of biological

and biomedical research has facilitated an in-

depth understanding of cellular and molecular

processes in health and disease. This has given

rise to the need for researchers to have access

to efficient high throughput services that are

too cost- and labor-intensive for single univer-

sity institutions. The Interdisciplinary Center for

Clinical Research (IZKF) Münster operates sev-

eral Core Units, which are a valuable resource

for an effective research environment.

Principal Aims

> The Core Units specialize in providing State-

of-the-Art skilled technical and instrument

support for the Medical Faculty of the Univer-

sity of Münster, extramural faculty, external

non-affiliated institutions as well as indus-

trial researchers, at affordable rates.

> Specialists in the field assist researchers

from the stage of project conception to its

completion thus ensuring maximum efficien-

cy.

> Researchers have open access to a large in-

strument pool consisting of expensive high-

end equipment that has increased the versa-

tile nature of the technology available to the

institutions of the Medical Faculty.

> The Core Units operate on a cost recovery ba-

sis and charges reflect the actual expenses

for consumables and service contracts.

Moreover, the Core Units promote translational

research by interacting closely with academia

and industry, ensuring that the technology

available is continuously updated. Lectures,

workshops and practical courses are organized

on a regular basis to keep users informed about

current developments.

This information folder provides an overview

of the different facilities, their technical equip-

ment and methodological support services

available to researchers.

Contact and more information

IZKF Scientific Office Albert-Schweitzer-Campus 1, Gebäude D3, 48149 Münster Tel.: +49 (251) 83-58695 Fax: +49 (251) 83-52946 Mail: [email protected]



>1

The IZKF Münster Technology Platform provides users with state-of-the-art ex-

pertise and equipment in four main technological areas, each of which consist

of several highly specialised Core Units (CU). The IZKF Scientific Office coordi-

nates and supports these Core Units and oversees their smooth operation.

Our information folder aims to provide users with an overview of the services

offered by the Core Units. For special experimental paradigms not listed, users

are requested to contact the Head of the respective Core Unit. It is manda-

tory for all users to strictly abide with the Terms of Use of the Core Units. The

IZKF Scientific Office provides information on prices and issues invoices for

services carried out. Users are advised to include costs for CU services in their

grant applications.

The logos on the upper right hand corner are specific to each CU, enabling

the easy identification of leaflets that have been updated. In addition, news

regarding novel technologies will be communicated to users via a newsletter.

As a user of our Technology Platform, we would appreciate your feedback on

our Technology Platform and urge you to contact the IZKF Scientific Office if you

have any concerns or constructive suggestions involving any of our facilities.



Integrated Functional Genomics

Small Animal Phenotyping

Transgenic Animal Models

Optical and Molecular Imaging

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The Core Unit Integrated Functional Genomics (IFG) is a multidisciplinary technology platform that pursues an integrated approach to understanding gene expression profiling and protein analysis in order to identify genes, proteins and peptides in complex samples. The bioinformatics unit uses mathematical, statistical and computing methods in the acquisition, stor-age, analysis and interpretation of data arising from genomic and proteomic projects. The ultimate aim is to relate gene and protein expression patterns to pathways, pathways to networks and networks to function in order to gain new insight into biological and disease circuits and develop therapeutic strat-egies. The automation of high-end molecular biology methods necessitates complex state-of-the-art instrumentation and technology. The IFG provides open access to this leading edge technology that is continuously validated according to the experimental needs of researchers, coordinates interdisci-plinary research projects and organises workshops. The IFG hosts the Annual Münster Conference on Single Cell and Molecule Analysis. This conference monitors the state-of-the-art in the field of nanoanalytics.

Transgenic animal models are a key tool to study the pathogenesis of various diseases and to test therapeutic strategies for human diseases. Since 1996 the IZKF supports the Core Unit TRAM, thus providing centralised services to all members of the Medical Faculty, WWU Münster at reduced costs.

Small animal phenotyping is the logical choice for the analysis of human cardiovascular diseases in mouse models. This platform consists of two units that characterise electrical activity and physiological function (CarTel) as well as ultrasound analysis of the murine cardiovascular system (ECHO).

The Optical and molecular imaging platform carries out non-invasive imag-ing techniques that aim to understand the molecular processes in small rodent models of human diseases at the intracellular level and in vivo by determining the bio-distribution of various radiolabelled tracer molecules (SmAP) and fluorochromes (OPTI). This platform was complemented by the CU Small animal MRI (SAMRI) in 2010, enabling High Throughput morpho-logical and functional MRI scans to be carried out.

IZKF Technology PlatformOverview

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The field of genomics emerged from the

initiation of various genome projects, and

the resulting knowledge created the field

of functional genomics, concerned with

analysing gene expression, epigenetics,

mutations and genetic variation, to name

a few. The Genomics facility offers its

customers a wide range of services from

single-cell microdissection, real-time

PCR, various microarrays to Next-Gener-

ation Sequencing (NGS). In addition to

providing the full service package from

experimental design to the final steps of

data interpretation, technical training is

offered to enable self-service handling of

the equipment.

Laser capture micro-dissectionLaser capture micro-

dissection (LCM) is

a unique technique

that enables RNA and

DNA to be isolated from pure specimens

of specific groups of cells or even single

cells devoid of contaminating adjacent

tissue. LCM can be performed on a vari-

ety of tissue samples including frozen tis-

sue sections and paraffin sections. Using

this method a sufficient number of cells

can also be collected from archival brain

tissue (Hasselblatt et al., 2006). This ma-

terial is pure enough to be used in down-

stream applications like PCR and genetic

fingerprinting. The P.A.L.M. Micro Laser

system is housed in a separate room and

is available to all users of the Genomics

unit.

Quantitative real-time PCRReal-time PCR continues to be the stand-

ard technology for gene expression stud-

ies. The 7900HT system (Applied Biosys-

tems) detects and quantitates nucleic

acids sequences in 384-well plates. This

system supports applications such as

gene expression, SNP genotyping, path-

ogen detection, viral load analysis and

miRNA quantification.

The 7900 HT system is available for self-

service after a demonstration by the

Genomics team.

TaqMan® Low Density ArraysThe 7900HT sys-

tem has an in-

terchangeable

block for TaqMan® Custom Array Cards.

These are 384-well micro fluidic cards

that enable 384 real-time PCR reactions

to be performed simultaneously without

the need for liquid handling robots.

Integrated Functional Genomics (IFG)

Genomics Facility

Laser capture microdissection workflow Courtesy of P.A.L.M. Microlaser Technologies

>4

This low- to medium- throughput micro

fluidic card allows for 1-8 samples to be

run in parallel against 12-384 TaqMan®

Gene Expression Assay targets that are

pre-loaded into each of the wells on

the card. The TaqMan® Custom Array is

completely customizable. Over 50,000

TaqMan® Gene Expression Assays de-

signed for human, mouse, rat genes, and

miRNAs are available. Additionally, pre-

configured TaqMan® Gene Signature Ar-

rays for human ABC transporters, human

angiogenesis, human apoptosis, human,

mouse or rat GPCR, human and mouse

Alzheimer, rat and human inflammation

etc. can be used.

Affymetrix microarraysAffymetrix arrays enable the simultane-

ous detection of mRNA abundance of

thousands of target genes to determine

whether they have been switched “on” or

“off” in a biological or pathological state.

The Affymetrix platform enables:

• DNA Copy Number Analysis

• Genome-Wide Genotyping

• Re-Sequencing Analysis

• Targeted Genotyping Analysis

• 3´IVT Expression Analysis

• Gene Regulation Analysis

• miRNA Analysis• Whole-Transcriptome Analysis

for many species like human, mouse, rat,

Arabidopsis, C. elegans, chicken, Dros-

ophila, E. coli, maize, rice, S. aureus,

Xenophus, yeast, zebrafish etc.

The Genomics unit offers a complete

gene expression analysis service from

controlling RNA integrity to supplying the

raw data. A basic or extensive analysis of

the data is carried out by the IFG bioinfor-

matics facility.

GenePix® microarray scanner

The GenePix® 4000B microarray scan-ner coupled with GenePix® Pro Micro-array Image Analysis Software and Acu-ity® microarray informatics software, sets the highest standards in the ac-quisition and analysis of data from all types of arrays, including nucleic acids, proteins, tissues, and cells. The Gene-Pix® 4000B scanner acquires data at user-selectable resolutions between 5–100 microns, allowing optimization of image resolution and file size for each experiment. The scanner features non-confocal optics, simultaneously 2-channel scanning with 532 nm and 635 nm lasers, and is usable for stand-ard microscope slides (e.g. Agilent mi-croarrays). It is available for self-service use.

Genepix 4000B Array-Scanner


Genomics Facility

Affymetrix GeneChip

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Next Generation Sequencing

The Next-Generation Sequencing (NGS) technology is the most up-to-date tech-nology available in genomic science. Thus, the IFG is proud to offer this tech-nology to its customers.

Our 5500xl SOLiD™ System (Applied Biosystems®/LifeTechnologies®) provides the combination of accuracy, sensitivity, and cost effectiveness to support large translational research studies. It helps to ensure optimal pro-ductivity with two flexible FlowChips, embedded quality controls, and project scalability. The system’s two configura-ble microfluidic FlowChips process up to 12 independent samples and the intelligent barcoding kits multiplex up to hundreds of samples in a single run. However, also projects with small sam-ple numbers can be run at a reasonable cost due to the flexible design. Pay-Per-Lane Sequencing and independent run lanes tailor the system to any project scale.

With the 5500xl Genetic Analyzer, the customer is empowered to discover rare genetic events or sub-populations of somatic mutations at an unprec-edented pace. The industry-leading ac-curacy of the 5500xl Genetic Analyzer enables detection of significant biolog-ical variation for applications for

Eukaryotes as well as Prokaryotes such as:

• Whole genome (Re-)Sequencing,

• Targeted Resequencing,

• SNP Detection,

• Structural Variant Detection,

• Whole Transcriptome Analysis,

• Gene Expression Analysis,

• Small RNA Analysis,

• Chromatin Immunoprecipitation Sequencing,

• Methylation Analysis

using

• Fragment Libraries (single or paired end) or

• Mate-paired Libraries

in

• Single-sample or

• Multiplex Design.

The NGS service provided by the IFG comprises the project design support, quality control (QC) of the customer-provided samples, library preparation, further QC steps, library quantification, sequencing run supervision, and vali-dation of the raw data. The raw data will be handed out to the customer who is free to use the IFG Bioinformatics ser-vice for secondary and tertiary analysis.

5500xl SOLiD™ System

Covaris S-series High Performance Sample Preparation System


Genomics Facility

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Bioinformatics support

The Genomics team works closely with the Bioinformatics team offering cus-tomers of the IFG professional scale

bioinformatics solutions. In addition

to standard services the bioinformatics

team supports specific requests with in-

dividual bioinformatics solutions tailored

to the specific needs of the customers.

Representative PublicationsSchmitz KJ, Helwig J, Bertram S, Sheu SY, Suttorp AC, Seggewiss J, Willscher E, Walz MK, Worm K, Schmid KW (2011) Differential expression of microRNA-675, microRNA-139-3p and microR-NA-335 in benign and malignant adrenocortical tumours. J Clin Pathol 64: 529-535.

Grundmeier M, Tuchscherr L, Brück M, Viemann D, Roth J, Willscher E, Becker K, Peters G, Löffler B (2010) Staphylococcal strains vary greatly in their ability to induce an inflammatory response in en-dothelial cells. J Infect Dis 201: 871-80.

Hasselblatt M, Mertsch S, Koos B et al. (2009) TWIST-1 is overexpressed in neoplastic choroid plexus epithelial cells and promotes proliferation and invasion. Cancer Res 69: 2219-2223.

Edemir B, Kurian SM, Eisenacher M et al. (2008) Activation of counter-regulatory mechanisms in a rat renal acute rejection model. BMC Genomics 9: 71.

Hasselblatt M, Bohm C, Tatenhorst L et al. (2006) Identification of novel diagnostic markers for choroid plexus tumors: A microarray-based ap-proach. Am J Surg Pathol 30: 66-74.

Dr. rer. nat. Jochen SeggewißIntegrated Functional Genomics (IFG)

Röntgenstraße 2148149 Münster

http://ifg-izkf.uni-muenster.de

Contact

Tel: +49 (251) 83 - 57168 Fax: +49 (251) 83 - [email protected] IZ

KF M

ünst

er (0

8/20

11)

WORKFLOW

Meeting with the Genomics & Bioinfor-matics team to discuss experimental de-sign, sample preparation and pricing

Researcher fills out the mandatory Order Form

- Sample submission

- Quality control of RNA / DNA

- Customer feedback

Microarray or High Throughput Sequenc-ing experiments are carried out

Raw or analysed data will be handed over to the customer as a hard copy


Genomics Facility

Please acknowledge the IFG Genomics / Bioinformatics Departments if the data generated by us is used in a publication and notify us when the manuscript is published

>7

Proteins are the principal effectors in

cellular function and the target of most

pharmacological strategies. The goal of

the proteomics facility is to provide cost-

effective access to sophisticated tech-

nology for modern proteomics analysis

in a central facility. Special emphasis is

placed on assisting researchers in de-

signing and evaluating their research

projects and includes extra features such

as advanced manual and bioinformatic

data mining.

Biomolecular mass spectromeryMass spectrometers are available (Q-TOF

Premier, ESI/AP-MALDI (IR/UV) ion trap,

MALDI-TOF), which can be flexibly used

for different types of experiments such as

the determination of molecular weights

of biomolecules regardless of their size.

Soft ionization techniques allow the anal-

ysis of intact proteins and labile modifi-

cations. Procedures have been devel-

oped to monitor enzymatic or chemical

reactions or to fingerprint biological flu-

ids. Not only proteins and peptides can

be investigated; the analysis of certain

types of drugs (e.g. Imatinib), fatty acids

or other substances in complex mixtures

is also possible.

Protein identificationThe analysis of proteins pre-separated

by electrophoretic or chromatographic

means is one of the main service tasks.

Reversed-phase nanochromatography

coupled to high-end mass spectrometry

(LC-MS; nanoUPLC/Q-TOF Premier) allows

confident assignment of proteins after

enzymatic digestion. Protein modifica-

tions such as phosphorylation, isoforms

and unknown sequences are accessible

in specially designed experiments.

Protein expression analysisThe The unit offers a choice of gel-based

or MS-based differential analysis. The

entire pipeline for the DiGE technol-

ogy (Differential Gel Electrophoresis) is

available. This is an advanced system

solution based on 2D-PAGE (separation

of proteins according to their isoelectric

point (pI) and molecular weight), which

enables the quantification of protein

expression profiles of two samples (e.g.

normal vs disease state) in a reproduci-

ble and statistically relevant manner. The

methodology eliminates gel-to-gel vari-

ance and represents the state-of-the-art

in gel-based expression analysis. DiGE

gels are scanned with the Typhoon 9400

three-laser scanner, which is also versa-

tile for use in other applications such as

modification-directed staining. Regulat-

ed proteins are subsequently identified

using LC-MS/MS.

A dedicated MS-based approach using Q-

TOF Premier MSE technology allows label-

free absolute and relative quantification

of proteins. It promises comprehensive

data with high reproducibility and re-

duced sample handling. Even less sam-

ple is needed (~2 µg compared to 500

µg for DiGE) and the data can be stored

indefinitely for later mining. The identi-

fication of hundreds of protein compo-

nents of one proteome is automatically

performed. The results of the analysis in-

clude not only the fold changes in regula-

tion but also provide clear insights about

the magnitude of proteome differences

and regulatory factors using principal

component analysis (PCA)- see Bioinfor-

matics section).


Proteomics Facility

>8

Protein separation and gel electro-phoresisThe unit assists in the preparation of tis-

sue homogenates or cell lysates. 1D- and

2D-electrophoresis can be performed

with customer samples. Moreover, the

fractionation of proteins in the liquid

phase based on their pI is possible, e.g.

to exclude dominant proteins for subpro-

teome analysis. Quality control of protein

separations is carried out with a minia-

turized capillary electrophoresis lab-on-

a-chip system. This method serves to rap-

idly diagnose separation procedures or

sample quality. Biological fluids such as

urine, cell extracts or whey can be evalu-

ated for their protein content.

Profiling of biofluidsLC-MS profiles are excellent means to

study biological changes. In particular,

urine profiles of low-to-medium molecu-

lar weight compounds have been shown

to reflect health or hormonal status. Rep-

licate samples are compared with statis-

tical tools such as PCA and the results are

evaluated further for biomarkers.

Protein arraysProtein arrays are increasingly used to

miniaturize interaction experiments.

They serve to detect proteins, study their

expression levels, as well as their interac-

tions and functions. Using protein arrays,

efficient and sensitive high-throughput

analysis of thousands of proteins can be

achieved simultaneously. Protein arrays

such as the FAST slides can be analysed

with two microarray scanners. Protein

arrays are commercially available for cy-

tokine and biomarker analysis as well as

autoimmune diagnostics.

Bioinformatics for proteomics and profilingIt has become increasingly necessary to

evaluate and visualize proteomic or mass

spectrometric data with statistical meth-

ods. A number of procedures have been

established for that purpose:

Protein expression analysis using DIGEThe DeCyder software applies a gel com-

parison method that introduces zero

statistical error, offering reliable data

and analysis. Co-detection, background

subtraction, normalization, and quanti-

fication of spots in images are improved

based on a warping function. The soft-

ware provides multivariate statistics

tools and enables the combined analysis

of different datasets, aiding the biologi-

cal interpretation of results by matching

with data retrieved from public and local

databases.

Pre-fractionation of protein mixtures for sub-proteome analysis


Proteomics Facility

>9

Protein expression analysis using LC-MSE

The expression module of ProteinLynx-

GlobalMiner 2.5 assists in the statistical

evaluation of LC-MS runs of proteome

digest replicate runs. Both relative and

absolute quantification is possible with-

out the need for sample labeling. Experi-

mental data are further mined using tools

such as Principal component analysis.

Principal component analysis (PCA)PCA (SimcaP, RapidMiner) as a linear

dimensionality reduction algorithm,

which projects high dimensional

data (replicate runs with hundreds to

thousands of data points) to lower

dimensional subspace spanned by

the principal eigenvectors and assist

investigators in finding major differences

in their samples. It is an important step

preceding biomarker assignment both

in proteomics and the analysis of LC-MS

profiles of urine or other biofluids

Exploratory analysis and data visu-alisationVisuMap software allows

understanding data that

otherwise resists easy in-

terpretation.

It provides a novel insight

into the patterns, relation-

ships and correlations be-

hind the data. In particu-

lar, relational perspective

mapping and advanced

clustering algorithms for

high dimensional data

like self-organizing map,

affinity propagation, k-

mean clustering are of

great value for analysis of

LC-MS data.

Pathway analysisThe information about regulated

proteins is further processed identifying

relationships, mechanisms, functions,

and pathways of relevance using

Ingenuity software. It delivers an

assessment of signaling and metabolic

pathways, molecular networks, and

biological processes that are most

significantly perturbed in the dataset of

interest.


Proteomics Facility

>10

Prof. Dr. rer. nat. Simone KönigIntegrated Functional Genomics (IFG)



Tel: +49 (251) 83 - 57164 Fax: +49 (251) 83 - 57255

[email protected]

Contact

IZKF

Mün

ster

(07/

2011

)

Protein Histidine Phosphatase (PHP)

PHP was discovered by the late

Prof. Susanne Klumpp and studied

extensively* in her group at the Institute

of Pharmaceutical and Medical Chemistry

at the WWU, Münster. As a result of

earlier collaborations, the IFG continues

to provide PHP for research purposes.

*Klumpp S, Krieglstein J (2009) Reversible Phosphorylation of Histidine Residues in Proteins

from Vertebrates. Sci Sig 10: 2(61):pe13

The following products are available on

request:

1. Dry recombinant PHP supplied in aliquots of 1 mg (or as required)

2. Glycerol culture of recombinant PHP supplied in aliquots of 1 ml (or as required)

Additional publications and further

information can be found at

http://php. uni-muenster.de

Representative PublicationsKummer MP, Hermes M, Hammerschmidt T et al. (2011) Nitration of Tyr10 critically enhances amyloid beta aggregation and plaque formation. Neuron. (In press)

Kriegeskorte A & König S, Sander G et al. (2011) Small colony variants of Staphylococcus aureus reveal distinct protein profiles. Proteomics 11: 2476-2490.

König S (2011) Urine molecular profiling distin-guishes health and disease: New ways in diag-nostics? Test case UPLC-MS. Exp Rev Mol Diagn 11: 383-391.

Wang W, Ackermann D, Mehlich A-M, König S (2010) False labelling due to quenching failure of N-hydroxysuccinimide-ester coupled dyes. Prot-eomics 7: 1525-2529.

Hasche A, Ferenz KB, Rose K, König S, Humpf H-U, Klumpp S, Krieglstein J (2010) Binding of ATP to nerve growth factor: Characterization and rele-vance for bioactivity. Neurochemistry Intern 56: 276-284.

Hohenester UM, Ludwig K, Krieglstein J, König S (2010) Stepchild phosphohistidine: Acid-labile phosphorylation becomes accessible by func-tional proteomics. Anal Bioanal Chem 8: 3209-3212.

Instrumentation such as array scan-ners, gel imagers and PAGE equipment are available for customer’s use.

Open access

Users can provide tissue, cells or pro-tein mixtures for analysis and are ad-vised to contact the proteomics staff who will be pleased to assist in ex-perimental design and to discuss the parameters that samples have to fulfill to ensure smooth processing and ex-cellent results.

Getting started

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Proteomics Facility

>11

The Bioinformatics Group provides cus-

tomers of the IFG with standard and

advanced statistical solutions for their

Microarray, Real-Time PCR and Next Gen-

eration Sequencing projects.

Especially the analysis of data from a

whole genome association study or a

NGS sequencing project requires pro-

fessional scale bioinformatics. We offer

standard assembly, alignment, mapping

genotyping and annotation services and

in addition we can support specific re-

quests with individual bioinformatics

solutions in close collaboration with cus-

tomers and scientists.

Next Generation Sequencing (NGS)The next-genera-

tion sequencing

(NGS) technolo-

gies constitute

various strate-

gies that rely on

a combination of template preparation,

sequencing and imaging, and genome

alignment and assembly methods. The

arrival of NGS technologies on the market

has changed the way we think about sci-

entific approaches in basic, applied and

clinical research.

We offer individual solutions for the anal-

ysis of NGS data from our Life technolo-

gies SOLiD 5500 System and other plat-

forms (e.g. Roche 454, Illumina HiSeq or

GA). We provide a NGS data service from

the primary basic analysis with quality

checks and base calling to the advanced

analysis with assembly, mapping, clus-

tering, annotation and visualization in

close collaboration with our customers.

NGS applicationsThe production of large numbers

of low-cost reads makes the NGS

platform useful for many applications.

These include variant discovery by

resequencing targeted regions of interest

or whole genomes, de novo assemblies of

bacterial and lower eukaryotic genomes,

cataloguing the transcriptomes of cells,

tissues and organisms (RNA–seq),

genome-wide profiling of epigenetic

marks and chromatin structure using

other seq-based methods (ChIP–seq)

and species classification and/or gene

discovery by metagenomics studies.

We use a flexible software pipeline ar-

chitecture based on Bioscope®, which

enables maximum flexibility and ease of

use for performing high throughput data

analysis for the SOLiD™ instrument data.

It provides the following features:

Alignment and MappingVarious bioinformatics tools are used

for the assem-

bly (Velvet) and

the alignment

(BFAST, BWA, Bowtie) and are intelligent-

ly linked up to form project-specific pipe-

lines. After the NGS raw data has been

generated, the color space reads are

aligned to a known reference sequence

or assembled de novo.

SNP detection

We use the diBayes software package

which allows sensitive and specific SNP

detection even at moderate to low cov-

erage. It al-

lows varying

the levels of

stringency and provides full control over

many filters for customization to fit par-

ticular experimental designs.


Bioinformatics Facility

01001011100010001010011010010001

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Structural variants detection All sequence variants other than single-

nucleotide variants, including insertions

or deletions, inversions, segmental du-

plications and copy-number differences

can be detected by the use of the special

Applied Biosystems software (AB Large

and Small InDel tools, Inversion and CNV

tools).

Whole transcriptome analysis

The Whole Transcriptome Analysis (WTA)

toolset (Bowtie, TopHat, and Cufflinks)

aligns the reads to a reference genome.

Using the mapping results, it counts the

number of tags aligned with exons, and

can convert the BAM file to the WIG for-

mat to view the coverage in a genome

browser (UCSC, IGV). The Expression

analysis includes statistical tests and the

calculation of p-values and fold changes.

WTA also supports experiments in fusion

detection. A fusion junction is a section

of transcribed RNA that maps to an exon

from one gene followed by an exon from

another gene. It occurs as the result of a

translocation, deletion, or chromosomal

inversion. A fusion junction excludes

exon- exon boundaries that arise from al-

ternative splicing for a gene.

The Affymetrix platformOur Affymetrix® Microarray technology

platform is a powerful tool for genomic

RNA and DNA analysis. Each study com-

prises multiple arrays with thousands of

transcripts or millions of markers (geno-

types, SNPs, CNV and

LOH marker) in groups

corresponding to differ-

ent experimental settings.

Conditions range from sin-

gle factor designs (treatments or tissue

types, times, benign-adverse in complex

diseases) with only a view arrays (includ-

ing technical and biological replicates) in

small groups up to whole genome associ-

ation studies with hundreds or thousands

of samples in a case-control design.

Primary and advanced analysisOur Bioinformatics facility provides users

with data from a primary analysis (qual-

ity, .CEL, .CHP, .CNT and exported text

and Excel-files) using the Affymetrix tools

(Genotyping and Expression Console,

GTC-Browser and Chromosome Analysis

Suite). In addition we provide results

and data from a more comprehensive

and complete microarray data analysis

in which we use the commercial Partek®

Genomics Suite, Ingenuity® Pathway

Analysis (IPA) and S-Plus (TIBCO® Spot-

fire S-Plus) scripts from our own software

development. For the advanced (second-

ary) analysis, which we coordinate in

close collaboration with our customers

we offer:

Expression analysisThis includes quality checks (background

adjustment, outlier detection), normali-

zation (standard, quantile and lowess

normalization), two group comparisons

to identify genes which show differential

expression (robust two sample t tests, F

tests comparing the mean

square of two varieties, fold

changes, Mann-Whitney-

Wilcoxon rank sum tests),

multiplicity adjustments for

the p-values (FWER: Holm-

Bonferroni, FDR: Benjamini-

Hochberg), multiple group

comparisons (ANOVA, basic

linear and nonlinear mod-

els and Neural Networks),

pattern discovery (hierar-

chical and model based

(two-way) clustering), principal compo-

01001011100010001010011010010001



>13

nents and factor analysis (Biplots, mul-

tidimensional scaling and gene shav-

ing), class prediction (linear discriminant

and nearest neighbors

analysis, classification

trees, Neural Networks,

risk analysis and ROC

curves) and annotation

with genomic informa-

tion including pathway analysis using the

IPA Pathway Analysis software.

Analysis of Whole Genome Associa-tion Studies (WGAS)WGAS are used in population genetics

to identify single nucleotide polymor-

phism (SNPs and their genotypes) which

can serve as marker sets in complex dis-

eases. A sufficient power requires a large

sample size, often 500 samples (arrays)

and more in each of two groups within a

case-control design. A typical WGA study

comprises three stages: screening (geno-

typing of ~106 SNPs using SNP 6.0 ar-

rays), replication of the

most promising SNPs in

matched cases and con-

trols and verification of

replicated association

signals in a larger co-

hort using an independent method (e.g.

TaqMan).

We provide full bioinformatics user sup-

port from the study design (sample size

and power calculations) during the whole

execution of the study (quality control

with reports from each array batch) to the

final genotype analysis of the TaqMan ex-

periments.

This includes quality checks (call rates,

identification of genotyping errors, Men-

del checks, tests for deviations from Har-

dy-Weinberg equilibrium and control of

minor allele frequencies), calculations of

Odds ratios (case vs. control) with FWER

and FDR control of the p-values for al-

lele and genotype frequencies in differ-

ent inheritance models, pairwise LD and

haplotype analysis, phenotype-genotype

association (susceptibility between

genotypes and disease) and relative risk

analysis using Logistic Regression (ad-

justed Odds ratios) and Neural Network

models.

Copy Number Variation (CNV) and Loss of Heterozygosity (LOH) analy-sisChromosomal abnormalities (allelic im-

balance, deletions, gains, Uniparental

Disomies (UPDs)) with structural changes

in the DNA play a major role in cancer

and cytogenetic re-

search. Amplification

of oncogenes and

loss of tumor sup-

pressor genes can

be detected by homozygous deletion or

loss of heterozygosity (LOH). Affymetrix

SNP 6.0 arrays with 906.600 SNPs and

946.000 CNV oligos and the Cytogenetics

Copy Number Assay (CytoScan HD) with

750.000 genotype-able SNPs and 1.8

million non-polymorphic probes allows

the user to perform a high-resolution

genome-wide analysis of chromosomal

aberrations.

Registered users can download the Chro-

mosome Analysis Sweet from Affymetrix

to analyze and compare the .cel files

from different experiments and to display

chromosomal abnormalities in a user-

friendly viewer.

We use Parteks Genomics Suite’s to de-

tect, display, and report regions of am-

plification or deletions on the genome.



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After identification of regions with signifi-

cant copy number changes (CNV) or LOH,

gene lists can be created and exported,

and corresponding gene or exon expres-

sion data can be seamlessly linked to the

mapping array data if available.

On user inquiry we develop special S-

Plus scripts e.g. to display LOH and CNV

regions from several case-control or tu-

mor vs. normal tissue experiments in one

(publication quality) graphic or to detect

significant marker regions by comparing

the discordant Chi-square distributions

(McNemar’s chi-square test) of markers

in case-control experiments.

Real-time PCR analysisWe carry out quality controls of the input

data to detect and filter errors that oc-

curred during the experiment. Embed-

ded methods permit endogenous genes,

which are optimal for normalisation, to

be determined. The applied software pro-

vides relative quantification analysis of

differential expression data, combining

interactive visualisation with advanced

statistics and data mining.

Representative PublicationsSchmitz KJ, Helwig J, Bertram S, Sheu SY, Suttorp AC, Seggewiss J, Willscher E, Walz MK, Worm K, Schmid KW (2011) Differential expression of microRNA-675, microRNA-139-3p and microR-NA-335 in benign and malignant adrenocortical tumours. J Clin Pathol 64: 529-535.

Grundmeier M, Tuchscherr L, Brück M et al. (2010) Staphylococcal strains vary greatly in their ability to induce an inflammatory response in en-dothelial cells. J Infect Dis. 201: 871-80.

Mees ST, Mardin WA, Wendel C et al. (2010) EP300--a miRNA-regulated metastasis suppres-sor gene in ductal adenocarcinomas of the pan-creas. Int J Cancer 126: 114-124.

Mees ST, Mardin WA, Sielker S et al. (2009) In-volvement of CD40 targeting miR-224 and miR-486 on the progression of pancreatic ductal ade-nocarcinomas. Ann Surg Oncol 16: 2339-2350.

Getting started

After consultation with the customer or scientist we perform a basic (prima-ry) or more comprehensive (second-ary) analysis of the raw data. Micro-array data (.CEL, .CHP, .CNT, .CSV and .XLS files) can be obtained on CDs or DVDs. The raw data from NGS experi-ments (.CSFASTA, .QUAL files) and the data from a secondary analysis (.BAM, .SAM, .GFF, .BAI, .BED and .CSFASTA.MA files) can be obtained on external 2TeraByte HardDisks. Alternatively all data can be downloaded from the IFG FTP-server.

Dr. rer. nat. Andreas HugeIntegrated Functional Genomics (IFG)


Dr. rer. nat. Reinhard VossIntegrated Functional Genomics (IFG)



Tel: +49 (251) 83 - 52207 Fax: +49 (251) 83 - 57255

[email protected]

Tel: +49 (251) 83 - 58964 Fax: +49 (251) 83 - 57255

[email protected]

IZKF

Mün

ster

(07/

2011

)

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TERMS OF USE

I. The Terms of Use of the Core Units of the IZKF Münster

serves to provide information for all users and to imple-

ment the statutes of the IZKF.

II. TheIZKFScientificOfficeactingonbehalfoftheBoardof

Directors is responsible for all administrative coordina-

tion.

1. TheCoreunitsshouldbeexclusivelyutilizedforresearch

purposes.

2. PriorityaccesswillbegiventoallmembersoftheIZKFand

theMedical Faculty.Theheadsof theCoreUnitsshould

ensure thatordersareprocessed ina timelymanner. In

ordertoenableoptimalplanningitismandatoryforinves-

tigatorstomakeanappointment.

3. UseoftheCoreUnitsbyresearchersfromotherfaculties,

universitiesorindustrialclientsisinvariablypossiblefol-

lowing submissionof awritten application. Cooperation

withotheruniversitiesandindustrialclientsnecessitates

thesigningofacontracte.g.MTAwiththeUKMorWWU

Münster, in order to protect the ownership rights of the

data acquired.

4. Membersof theMedicalFacultywillbecharged forcon-

sumables.MembersofotherNaturalSciencesFacultiesof

theWWUMünsterwillbechargedadditionalcosts(partial

costrecoveryexpensesforservicecontracts).Allexternal

usershavetopayservicefeesincludingvalue-addedtax

(VAT),asoutlinedintherespectivepricelists.

5. Scientificcooperationbetweentheheadofafacilityand

otherscientistsissolelyintendedfortheadvancementof

methodsand technology. In exceptional cases it is pos-

sibletocarryoutascientificcooperationthatexceedsthe

routine service character. These exceptions have to be

justifiedintheapplicationandrequirethepermissionof

theIZKFBoardofDirectors.Costsforconsumableshaveto

bereimbursed.Additionaluserchargesdonotapply.All

publicationsresultingfromascientificcooperationshould

acknowledgetheIZKFMünsteronthetitlepageunderthe

authors’ address.

6. Inthiscontextitisnotpossibletoconsiderroutineinvesti-

gationsasascientificcooperationandtheseservicesare

liableforfees.

7. Allmembersoftheresearchgroupsareresponsibleforthe

correctoperationandhandlingoftheequipment.General

labsafetyruleshavetobeobserved.

8. Theequipmentbelongingtothe IZKFMünstercannotbe

relocated. Experiment-based relocation of equipment is

onlypossibleafterobtainingpriorconsent fromthe IZKF

ScientificOffice.

9. TheresearchersoftheCoreUnitsareobligatedtohandle

theresultsanddatainastrictlyconfidentialmanner,and

cannot duplicate, publish or use the data for any other

purposes.Exceptionstothisrulehavetobedocumented

inwriting.

10. UsersoftheservicesaffirmthattheCoreUnitsoftheIZKF

Münstertakenoresponsibilityforcontaminatedsamples.

Usersalso takecomplete responsibility foralldata/ re-

sultswithrespecttosafety,completenessandconfidenti-

ality.

TheseTermsofUsewerepassedbytheIZKFBoardofDirec-

torson06.November2007andtakeeffectimmediately.

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Documents

ChronicDisease - uni-muenster.decampus.uni-muenster.de/fileadmin/einrichtung/ifg/IFG_Broschuere.pdf · This information folder provides an overview of the different facilities, their