genomics and proteomics core facility (gpcf) seminar series · page 2 genomics & proteomics...

Genomics and Proteomics Core Facility (GPCF)

Seminar Series

16.07.2007, DKFZ, INF 280, Lecture Hall H1

Genomics & Proteomics Core Facility Objectives

• State-of-the-art Services and Technologies• Service at highest Quality• Ensure short Turn-around Time• Equal Access• Dedicated Personel and Responsibility for Service Duties• Education• Discriminate between Service and Research• Capacity Utilization / Investment Cost / Efficiency• Fee for Service• Annual User Review

• Scientific Infrastructure (W1xx)• CF Committee (WiRa)

Genomics & Proteomics Core Facility Structure of Core Facilites

• Animal Care (N. Howells)• Animal Housing• Transgenics• Mouse Models• ...

• IT Services (H. Haas)• Central Computing Facility• Networks• Software Support• ...

• Imaging (N. N.)• Confocal Microscopy• Electron Microscopy• Cell Sorting/FACS• ...

• Library Services (H. Metzler)• ...

• Genomics & Proteomics (B. Korn)• ....

Many Units 5 Core Facilities

Genomics & Proteomics Core Facility Ways to provide service

• Full service• Order of user, return of product/information (e.g. peptide,

expression profile...)• Supervised access

• Central investment and expertise provided, but user iskey for work (e.g. 2D gels, Real-time PCR)

• Trans-center service• One center provides established servcie for

another/multiple other centers (e.g. chemical screening service by Joe Lewis (EMBL/DKFZ)

• Public-private service• Service provided together with private company (e.g.

sample preparation in-house, external service, analysisin-house (Rahmenvertrag Protagen))

• Outsourcing

Genomics & Proteomics Core Facility Program

• Genomics• Sanger Sequencing (A. Hunziker)• “Next Generation” Sequencing (G. Nyakatura)• Expression Profiling (B. Korn)• Genotyping (B. Korn)• Clone Repository (B. Korn)

• Proteomics & Structure Analysis• Peptide synthesis (R. Pipkorn)• Protein Interaction Screening (M. Kögl)• Protein Analysis, 2D (T. Kempf)• Protein Analysis MALDI, esiMS/MS (M. Schnölzer)• Mass Spectrometry: Small molecules and protein

modifications (W. Lehmann)• NMR (W. Hull)• Modeling (C.W. v. d. Lieth)

• Meet People of your Service Units (Foyer)• Informal meeting for more detailed questions

Sanger SequencingAndreas Hunziker

Genomics & Proteomics Core Facilities, DKFZ

Genomics & Proteomics Core Facility We offer

• DNA-sequencing from purified • Plasmids and • PCR products

• We provide more than 100 vector primers

Genomics & Proteomics Core Facility Read-length

Our sequencing reaction read-lengths are normally between 600 and 800 bases and starts 1 to 20 bases after the primer.

It depends upon several factors: • template concentration• purity• base composition poly A and/or T regions

Genomics & Proteomics Core Facility

GC content and repeats


Turn-around Time• 1-5 working days for easy single runs (if the

primer is available).• You may be able to get the fastest turnaround

time by bringing the samples before 9:30 AM.

What we need• The submission form.For a single run• 300ng Plasmid DNA, conc. >40ng/µl• 10ng/100 bases PCR product.• Customer primer >10µM


What you get• Normally we send the sequence as a text file by

Email. When desired, you can also get the chromatogram.

Cost of Service• The basic cost of DNA sequencing for processing

one template with one primer is • € 5 for raw data, • € 10 for an edited sequence and• € 5 for a new designed primer


Contact Info:The sequencing Facility is located at DKFZ-Heidelberg, INF580, room B1.306.

For information about sequencing and ordersplease contact:

Andreas HunzikerEmail: [email protected]: 42 46 69

Next Generation Sequencing Gerald Nyakatura


Genomics & Proteomics Core Facility Overview: Next Generation Sequencing

• Contact• Gerald Nyakatura ([email protected], -3409)

• Offer• The high throughput sequencing unit provides highly

parallel DNA sequencing service based on the 454/Roche GS20FLX technology. This technology coined “nextgeneration sequencing” enables the generation of up to 100 Mb of sequencing information in a single run.

• Assistance in experimental design• Sample preparation starting from customer prepared DNA• Sequence analysis on the Genome Sequencer 20• Bioinformatics support for the analysis of resulting

sequence data

Genomics & Proteomics Core Facility Next Generation Sequencing: Performance

• 500 Fold Throughput and Cost Advantage over Fluoresence Sequencing

• Sequencing of up to 100 Mb per 8 hour instrument run

• Up to 400 000 sequence reads with average length in excess of 200 bp

• Consensus accuracy in excess 99.99% at 10-15 fold over-sampling


Next Generation Sequencing: 454/Roche Workflow





sequence data

Genomics & Proteomics Core Facility Second Generation Sequencing: emPCR

•http://www.dkfz.de/gpcf

• More Information on Technology• Contact Form• Detailed Applications List• Links to Relevant Literature

Genomics & Proteomics Core Facility Second Generation Sequencing: Sequencing




Second Generation Sequencing: Pico Titer Plate



Genomics & Proteomics Core Facility Second Generation Sequencing: Sequencing



Genomics & Proteomics Core Facility Next Generation Sequencing: Applications





sequence data

Genomics & Proteomics Core Facility Summary: Second Generation Sequencing

• Personell

• Gerald Nyakatura• Anne Arens (Bioinformatics)• André Leischwitz (Technical Assistance)

• Turn-around Time• From 2 Weeks (depending on project)

• Quality• Input: From 10 ng• Quality control at all levels• Assistance in Planning of Experiment

• Cost• Starting at ~Euro 5000

Genomics & Proteomics Core Facility Second Generation Sequencing: Webpage



Expression ProfilingBernhard Korn


Genomics & Proteomics Core Facility Overview: Expression Profiling

• Contact• B. Korn ([email protected], -4700)

• Offer• Whole Genome profiling

• Illumina (human, mouse, rat; ~750 ng total RNA required)• Affymetrix (wide species portfolio, exon chips; 5 µg total RNA

required)• Small comparison projects (list of regulated genes)• Large scale studies (complex analysis, gene signatures)

• Support in experimental set-up, basic data analysis• Courses on expression profiling and analysis, central software

support• Outlook (08/07)

• Expression profiling on defined gene sets (<1.500 genes) with high sample throughput

• Profiling on formalin-fixed tissue• Methylation Analysis

Genomics & Proteomics Core Facility Affymetrix

• 25mers• 11-20 / gene• 46.000 clusters• PM / MM

Genomics & Proteomics Core Facility Illumina

• 50mers• 1 / transcript• av. >30 replicates• 23.00 - 48.000 clusters• 800 – 7200 ctrls• 1,7 mio beads (statistics)

Genomics & Proteomics Core Facility BeadChip® and GeneChip® Systems

SoftwareData Analysis

FluidicsStation

Chip Scanner

Illumina

Affymetrix

Genomics & Proteomics Core Facility Summary: Expression Profiling

• Personal• Sabine Henze• Oliver Heil

• Turn-around Time• 2-3 weeks

• Quality• Quality control at all levels (total RNA, labeling, fragmentation,

hybridization, scanning, analysis)• Data Return

• QC Data, Raw Expression Data• Interpreted Data (MS Office compatible)• Personal Meeting

• Cost• Starting at 250€ / whole genome profile

Genomics & Proteomics Core Facility Expression Profiling Webpage

www.dkfz.de/gpcf

submissionform

GenotypingBernhard Korn


Genomics & Proteomics Core Facility Overview: Genotyping

• Contact• B. Korn ([email protected], -4700)

• Offer• Whole genome analysis, using Infinium Technology

(370k, 550k, 650k, 1m SNPs)• Genotyping• Copy number variations• Linkage• LOH

• Customized SNP set screening, using GoldenGate Technology• <1536 markers• >384 samples

• Basic support for data analysis• Outlook (10/07 and 07/08)

• Partial automation of GoldenGate• Clinical diagnostics, counseling (Dept. Human Genetics, Univ. HD)

Genomics & Proteomics Core Facility GPCF: Infinium Technology I

Genomic DNA amplification (WGA)750 ng

Fragmented DNA >10 µg

Hybridize DNA to beads on chip

Select at high stringency

Genomics & Proteomics Core Facility GPCF: Infinium Technology II

Single base extension,depending on allele

av. >20 replicates

Beadchip: scanning is 15-45 minper genome

Genomics & Proteomics Core Facility GPCF: Infinium Analysis II

HL-60Hemizygous Del

MCF-7Homozygous andHemizygous Del

3X female

Genomics & Proteomics Core Facility GPCF: GoldenGate Assays

• Customized genome analysis (96-1536 marker/SNPs)• Genotyping• Expression • Methylation• High throughput (multiples of 96 patients)

• Quality• Input DNA QC and conc. (picogreen)• Labeling, hybridization and scanning controls• Call rates >97-98 % (depending on selected SNP set)• Basic analysis (on-board tools)

• Status• Pilot projects since 05/07• First large scale screen (4.000 patients)

• Outlook• Up to 12.000 samples per year• Validation of established marker sets and signatures

Genomics & Proteomics Core Facility GPCF: GoldenGate Technology

Genomic DNA (>250 ng)

Activate and add primers(3 oligos per SNP)

Extension and ligation

Universal PCR

Genomics & Proteomics Core Facility GPCF: GoldenGate Technology

Hybridize address code toBeadarray

SAM Beadarray (96 samples)

Image of pin(1 sample, 1536 SNPs, av. 30 replications)

address code

Genomics & Proteomics Core Facility Summary: Genotyping

• Personal• Matthias Schick (GoldenGate)• Sabine Henze (Infinium)• (Oliver Heil)

• Turn-around Time• 2-3 Weeks (Infinium)• 1 to >4 Months (GoldenGate)

• Quality• Input 500-750 ng genomics DNA (50 ng/µl, Picogreen)• Quality control at all levels (WGA, labeling, hybridization,

scanning, analysis)• Call Rates: Infinium (>99%), GoldenGate (>97%)

• Data Return• Raw Data• Interpreted Data (MS Office compatible)• Personal Meeting

• Cost• Starting at ~700€ / 2x 370k marker Infinium analysis

Genomics & Proteomics Core Facility Genotyping Webpage

www.dkfz.de/gpcf

Clone & Vector RepositoryAndré Leischwitz


Genomics & Proteomics Core Facility Overview: Clone Repository

• Contact• A. Leischwitz ([email protected], -4734)

• Offer• Full Open Reading Frame (ORF) Clones

• Gateway format, with/without stop codon• Fully sequenced (majority in EMBL/Genbank)• >7.000 clones (mainly human)• Search interface (Keyword, IDs, blast)• Collab. Harvard Institute of Proteomics

• Outlook (09/07)• Vector repository

• Commercial and home-made vectors• E.coli strains

Genomics & Proteomics Core Facility How to obtain Clones

1. Login at www.dkfz.de/gpcf2. Navigate to

“Vector & Clone Repository”3. Open “Search for

Gateway Full ORF clones“4. Enter Query


keywordsIDs (e.g. AccNo, unigene)symbolsfasta sequences

1. Login at www.dkfz.de/gpcf2. Navigate to

“Vector & Clone Repository”3. Open “Search for

Gateway Full ORF clones“4. Enter Query


5. Check results6. Verify information7. “Order overview”8. “Submit”

Genomics & Proteomics Core Facility Summary: Clone Repository

• Turn-around Time• <1 Week

• Quality• All clones fully sequenced (error rate <1/104)• Constantly extended Collection• Biannual Annotation update

• Material/Information Return• E.coli strain (kanR), to be picked up INF515• Full Manual available for download• Complete Sequence, incl. Vector transitions

• Cost• 30€

• Limitations• No transfer to collaborators outside of DKFZ

genomics and proteomics core facility (gpcf) seminar series · page 2 genomics & proteomics...

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