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Genomics and Proteomics Core Facility (GPCF)
Seminar Series
16.07.2007, DKFZ, INF 280, Lecture Hall H1
Page 2
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)
Page 3
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
Page 4
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
Page 5
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
Page 7
Genomics & Proteomics Core Facility We offer
• DNA-sequencing from purified • Plasmids and • PCR products
• We provide more than 100 vector primers
Page 8
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
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Genomics & Proteomics Core Facility
GC content and repeats
Page 10
Genomics & Proteomics Core Facility
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
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Genomics & Proteomics Core Facility
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
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Genomics & Proteomics Core Facility
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 Facilities, DKFZ
Page 14
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
Page 15
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
Page 16
Genomics & Proteomics Core Facility
Next Generation Sequencing: 454/Roche Workflow
• 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
Page 17
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
Page 18
Genomics & Proteomics Core Facility Second Generation Sequencing: Sequencing
•http://www.dkfz.de/gpcf
• More Information on Technology• Contact Form• Detailed Applications List• Links to Relevant Literature
Page 19
Genomics & Proteomics Core Facility
Second Generation Sequencing: Pico Titer Plate
•http://www.dkfz.de/gpcf
• More Information on Technology• Contact Form• Detailed Applications List• Links to Relevant Literature
Page 20
Genomics & Proteomics Core Facility Second Generation Sequencing: Sequencing
•http://www.dkfz.de/gpcf
• More Information on Technology• Contact Form• Detailed Applications List• Links to Relevant Literature
Page 21
Genomics & Proteomics Core Facility Next Generation Sequencing: Applications
• 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
Page 22
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
Page 23
Genomics & Proteomics Core Facility Second Generation Sequencing: Webpage
•http://www.dkfz.de/gpcf
• More Information on Technology• Contact Form• Detailed Applications List• Links to Relevant Literature
Expression ProfilingBernhard Korn
Genomics & Proteomics Core Facilities, DKFZ
Page 25
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
Page 26
Genomics & Proteomics Core Facility Affymetrix
• 25mers• 11-20 / gene• 46.000 clusters• PM / MM
Page 27
Genomics & Proteomics Core Facility Illumina
• 50mers• 1 / transcript• av. >30 replicates• 23.00 - 48.000 clusters• 800 – 7200 ctrls• 1,7 mio beads (statistics)
Page 28
Genomics & Proteomics Core Facility BeadChip® and GeneChip® Systems
SoftwareData Analysis
FluidicsStation
Chip Scanner
Illumina
Affymetrix
Page 29
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
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Genomics & Proteomics Core Facility Expression Profiling Webpage
www.dkfz.de/gpcf
submissionform
GenotypingBernhard Korn
Genomics & Proteomics Core Facilities, DKFZ
Page 32
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)
Page 33
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
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Genomics & Proteomics Core Facility GPCF: Infinium Technology II
Single base extension,depending on allele
av. >20 replicates
Beadchip: scanning is 15-45 minper genome
Page 35
Genomics & Proteomics Core Facility GPCF: Infinium Analysis II
HL-60Hemizygous Del
MCF-7Homozygous andHemizygous Del
3X female
Page 36
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
Page 37
Genomics & Proteomics Core Facility GPCF: GoldenGate Technology
Genomic DNA (>250 ng)
Activate and add primers(3 oligos per SNP)
Extension and ligation
Universal PCR
Page 38
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
Page 39
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
Page 40
Genomics & Proteomics Core Facility Genotyping Webpage
www.dkfz.de/gpcf
Clone & Vector RepositoryAndré Leischwitz
Genomics & Proteomics Core Facilities, DKFZ
Page 42
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
Page 43
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
Page 44
Genomics & Proteomics Core Facility How to obtain Clones
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
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Genomics & Proteomics Core Facility How to obtain Clones
5. Check results6. Verify information7. “Order overview”8. “Submit”
Page 46
Genomics & Proteomics Core Facility How to obtain Clones
5. Check results6. Verify information7. “Order overview”8. “Submit”
Page 47
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