GSU

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GSU

• Genetic Service Unit

02/02/2007 - presented by Paul Coucke Jan
Hellemans
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• Zware impulsfinanciering 2004 University Ghent
• (promotor Prof. A. De Paepe)
• Sequencing and genotyping facility
• Reasons
• Increasing demand of our own genetic department
• Requests from many other UGent laboratories
• Increased efficiency (both in performance and
  price)
• - High-through put DNA sequencing more and more
  important in molecular biology and very powerful
  technique to analyse genes
• - High-through put SNP analysis and quantitative
  PCR technology state of the art technique in
  modern research

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Overview

• General information
• Overview of all equipment in the GSU
• Overview of the software applied in the GSU
• Overview of the available services
• Concluding remarks

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Website
http//medgen.ugent.be/CMGG gt faciliteiten
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Contact us

• Address
• Medical Research Building
• UZ-Gent
• De Pintelaan 185, 9000 Gent
• E-mail
• Info.gsu_at_medgen.ugent.be
• Tel
• 09 240 46 71

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Equipment

• DNA isolation robot
• Autopure LS, Gentra, Qiagen
• Thermocyclers
• 2x384 well
• Sequencers
• ABI3130xl
• ABI3730xl
• Pipetting robots
• Sciclone ALH3000
• Sciclone ALH4000
• qPCR machines
• Icycler
• LightCycler 480

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Software

• Finch
• On-line request and sequence data management
  system
• Run-editor (under construction)
• Allows run annotation to be completed remotely
• SeqScape
• Automated alignment of sequences against a
  reference sequence
• Ideal for resequencing
• GeneMapper
• Automated genotyping of microsatellites (and
  SNPs)
• qBase
• Automated calculation of relative quantities from
  Ct values, supporting normalization with multiple
  reference genes and inter-run calibration
• Progeny
• Drawing pedrigrees and haplotypes

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Services

• Practical services
• DNA isolation
• DNA sequencing
• Microsatellite analysis
• SNPlex (soon available)
• Real-time quantitative PCR (qPCR)
• 2. Theoretical services
• Technical theoretical information
• Help with experiment design
• Practical information (e.g. standard protocols)

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• DNA isolation

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DNA isolation - equipment

• Autopure LS, Gentra, Qiagen

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DNA isolation theoretical information

• Puregene Genomic DNA Purification Kit
• salt-extraction (http//www.gentra.com).
• Step 1 Lysis of red blood cells and removal of
  the lysate
• Step 2 Lysis of white blood cells with anionic
  detergents in the presence of a DNA stabilising
  agents
• Step 3 Removal of proteins by salt-precipitation
  DNA remains in solution
• Step 4 DNA precipitation (ethanol based)DNA is
  dissolved in TE buffer

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DNA isolation technical information

• Fully automated system
• Process either 8 or 16 samples at one time
• 16 samples in 1h 20min 96 samples in 8 hours
• 1 10 ml whole EDTA-blood (not validated for
  other samples types yet)
• Puregene chemistry (300 µg DNA from 10 ml blood)
• No system available to isolated small blood
  volumes

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DNA isolation practical information

• Announce samples by email (gt10)
• Samples in a study (on demand)
• Unique identifier necessary for each sample
• One week service
• Average yield 30 µg/ml
• Cost 16 Euro for 1-10 ml EDTA blood
• No storage, no DNA concentration measurement

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• DNA sequencing

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DNA sequencing - equipment
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DNA sequencing theoretical information

• Raw PCR product Sequencing primers

PCR reformatting PCR cleanup (enzymatic)
Purified PCR product Primer
Seqencing reaction (Big dye terminator v3.1)
Raw sequencing product
Sequence cleanup (Agencourt magnetic beads)
Purified sequencing product
96 Capillary sequencer ABI3730xl
DNA sequence
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DNA sequencing - options

• Option 1
• Sequencing run
• 2.50 / sequence
• Requires 10µl sequence product (cleaned up)
• Results in 2 days for full plates, 2 to 5 days
  for partial plates
• Option 2
• PCR clean-up sequencing reaction clean-up
  sequencing run
• 4.00 / sequence
• Requires
• PCR product 10µl
• Primer 5µl 1.5µl per reaction(nothing in case
  of standard primers, e.g. M13)
• Results in 2 to 5 days

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DNA sequencing - workflow

• Make a request in Finch

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DNA sequencing - workflow

• Make a request in Finch
• Deliver material to MRB
• Order (printed from SAP)
• PCR products
• Primers
• 0 boxes (standard primers)
• 1 box (unidirectional seq)
• 2 boxes (bidirectional seq)

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DNA sequencing - workflow

• Make a request in Finch
• Deliver material to MRB
• Combine requests into runs and prepare robot work
  lists (script Finch)
• Combine PCR products into full 96-well plates
  (ALH4000)
• Enzymatic PCR clean-up (ALH4000 thermocycler)
• Prepare and perform 5µl sequencing reactions in
  384-well plates (ALH3000 thermocycler)
• Sequencing clean-up with magnetic beads (ALH4000)
• Sequence run (ABI3730xl)

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DNA sequencing - workflow

• Retrieve results from Finch

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DNA sequencing - results

• Long run (2 hours)
• QV 30-55
• QV20 800bp
• Standard run (1 hour)
• QV 30-55
• QV20 650bp

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• Microsatellite analysis

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Fragment analysis - equipment
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Microsatellite analysis

• Supported dye sets
• DS-30, filter set D 6-FAM, HEX, NED, ROX (size
  standard)
• DS-33, filter set G5 6-FAM, VIC, NED, PET, LIZ
  (size standard)
• Supported formats
• 16-well _at_ ABI3130xl (25 / 16)
• 96-well _at_ ABI3730xl (150 / 96)
• Requires 96-well plate with 16 or 96 wells filled
  with
• 10µl high purity formamide
• appropriate size standard
• adjusted amount of fluorescently labeled PCR
  product
• Results in 2 days
• Standard protocol for custom microsatellites on
  CMGG website (http//medgen.ugent.be/CMGG/onderzoe
  k.php?topic_id11)
• Genome wide linkage analysis kits available

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• SNPlex
• Soon available

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SNPlex - equipment
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Comparison of genotyping technologies

• Few SNPs (e.g. 3)
• TaqMan
• Melt-curve analysis
• MS
• Average number of SNPs (25 - few hundred)
• SNPlexNot suited for the analysis of few samples
  since probes are ordered in sets for 5000 samples
• Many SNPs (gt1000)
• Beads
• Arrays
• Due to price constraints sample number is usually
  limited

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SNPlex theortical information

• High throughput SNP genotyping based on OLA/PCR
  reactions

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SNPlex technical information

• Reagents
• Select SNPs
• ABI designs and provides probe pools for these
  SNPs
• Pool properties
• Ideal size multiple of 48
• Provide an excess of SNPs to account for dropout
  in multiplexing
• Current capacity 5000 genotypes / day
• Price depends on number of SNPs and samples
• 0.10 - 0.50 / genotype
• Inform for more details

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• qPCR

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qPCR - equipment
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qPCR - options

• Option 1
• Just qPCR run
• 25.00 / 384-well plate
• Option 2 (soon available)
• qPCR (SYBRgreen) pipetting qPCR run
• 125.00 - 200.00 / 384-well plate (depending on
  mastermix)
• Option 3 (soon available)
• Experiment design primer optimization qPCR
  pipetting qPCR run
• Inform for pricing
• Option 4 (soon available)
• Collaborations including everything from option 3
  data analysis

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qPCR practical information
Run-editor

• Select plate format and application
• Fill in run annotation
• Export run information and send it to GSU

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qPCR practical information

• 384-well plates
• 1-4 quadrants filled
• Small volumes
• ? Lower prices
• Experiment design 1 (reservoir)
• 1 target per quadrant
• 1 target 384 samples4 target 96 samples
• Experiment design 2 (strips)
• 1 target per quadrant and per row
• Fill DNA in plates according to primers in strips
• Max 32 genes (for 12 samples)

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qPCR - results

• Reproducibility (96 times same mastermix and DNA)
• Stdev(cycle) 0.086
• Max difference(cycle) 0.49
• CV(relative quantity) 6.10

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Practical financial information

• Prepare an internal order in SAP system
• Supplier number GE02 (Vakgroep Pediatrie en
  Genetica)
• Article number 3000038 (int. Wetenschappelijke
  dienst)
• Order number style 4 3XX XXX XXX
• Include service or product code in order
• A printout of an approved internal order is
  required for every service or product
• Available products
• SNPlex buffers - qPCR reagents LCGreenPLUS

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General remarks

• Quality and time management
• If complaints, please contact us!
• Prices
• ISO17025 (in preparation)
• DNA-extraction
• DNA-sequencing
• Advantages
• Automation
• Small reaction volumes
• Purchase of products in large volumes
• Advise help in experimental design

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• Departmental head
• Anne De Paepe
• Laboratory for Molecular Genetics head
• Paul Coucke
• Genetic Service Unit supervisor
• Jan Hellemans
• Scientific advisor qPCR
• Jo Vandesompele
• Genetic Service Unit operator
• Renée Dekens
• Inge Vereecke
• Petra Vanacker
• Elien Imant
• Contact
• Info.gsu_at_medgen.ugent.be
• 09 240 46 71