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Contract Research - Frequently Asked Questions
        FAQs are broken down by contract research type. Sometimes it is worth looking at related areas to gain further information.



DNA Extraction

Question Answer
What method do you use to extract the DNA?
 We currently out-source DNA extraction to our experienced ISO 9001 certified European partner, their extraction chemistry is based on magnetic bead technology. The magnetic separation eliminates centrifugation and phenol extraction steps and allows us to fully automate the whole process. There are three basic steps: the first step involves the lysis of cells and binding of DNA to the magnetic particles. The DNA is then washed three times and eluted.
What is the typical DNA yield from a 10ml blood extraction?
 The yield is dependent on the amount of DNA containing cells in the sample but is typically 300-500ug of DNA from a 10ml blood extraction, 15-25ug from a 2ml blood extraction, 4-6ug from a 200ul blood extraction.
Are you able to extract DNA from small volume of blood?

 Yes, we can extract DNA from as little as 10ul of blood. A minimum of 20 samples is required for small volume (less than 2ml blood) extraction.
Are you able to extract DNA from buccal swabs/FTA paper?

 Yes, our extraction method is able to extract high quality DNA from buccal swabs and FTA paper provided the swabs and paper have been stored appropriately according to the manufacture's instructions.
Are you able to extract DNA from cell lines?

 Yes, our extraction method is able to extract high quality DNA from frozen cell pellet.
What is the quality and stability of the extracted DNA?

 The extraction methods produce purified PCR-ready genomic DNA. The typical ratio of absorbance at 260nm and 280nm of the extracted DNA is between 1.7 and 1.9 and the expected size the extracted DNA is more than 20Kb. Typical results from using the extraction method are shown in the following figures.

Table 1: Yields of DNA extracted from different starting materials

Sample ID: Concentration DNA (ug/ul) Total yield (μg) Ratio A260/A280 Sample material
1 0.045 9.0 1.77 1ml Whole blood
2 0.067 13.4 1.78 1ml Whole blood
3 0.011 2.2 1.89 200μl Buffy Coat
4 0.050 10.0 1,79 200μl Buffy Coat
5 0.048 9,6 1,78 1ml Whole blood
6 0.047 9.4 1.78 1ml Whole blood
7 0.069 13.8 1.81 1ml Whole blood
8 0.045 9.0 1.83 1ml Whole blood
9 0.032 6.4 1.80 1ml Whole blood
10 0.039 7.8 1.82 1ml Whole blood
11 0.054 10.8 1.75 1ml Whole blood
12 0.077 15.4 1.82 1ml Whole blood
13 0.041 8.2 1.83 1ml Whole blood
14 0.041 8.2 1.81 1ml Whole blood
15 0.043 8.6 1.84 1ml Whole blood
16 0.038 7.6 1.74 1ml Whole blood
17 0.062 12.4 1.81 1ml Whole blood
18 0.026 5.2 1.83 1ml Whole blood
19 0,034 6.8 1.87 1ml Whole blood
20 0.054 10.8 1.89 1ml Whole blood
21 0.028 5.6 1.78 1ml Whole blood
22 0.109 21.8 1.53 1ml Whole blood
23 0.032 6.4 1.88 1ml Whole blood
24 0.083 16.6 1.82 1ml Whole blood
25 0.036 7.2 1.80 200μl Buffy Coat
26 0.022 4.4 1.89 200μl Buffy Coat
27 0.114 45.6 1.84 200μl Cultured cells
28 0.162 32.4 1.83 200μl Cultured cells
29 0.087 17.4 1.82 200μl Cultured cells
30 0.245 49.0 1.65 200μl Cultured cells
31 0.041 8.2 1.83 200μl Cultured cells
32 0.074 14.2 1.82 200μl Cultured cells


Figure 1: The reproducibility of our extraction chemistry on automated liquid handling systems. Each sample was extracted in triplicate to show that the yield of each sample is similar and high molecular weight DNA is obtained.



Figure 2: A cross contamination test. Blood samples and water were arranged alternatively in automated liquid handling system for extraction. After extraction, PCR reactions amplifying a fragment of the CFTR gene (exon 4) were set up using the eluate of all the samples. PCR product was only detected from the eluates from the blood samples and not the water.

insert fig 2 image
What is the stability of the extracted DNA?

 The genomic DNA is stable long term when buffered in Tris, pH 8 and stored at -20 degrees C.
How should I send the samples to Geneservice?
 Recommended shipping conditions for various sample types are as follows:
  • Cells, dry ice

  • Blood, dry ice

  • Buccal swabs, dried at room temperature

  • FTA paper, dried at room temperature.

    • It is essential that in addition to a signed agreement and purchase order number that we also get the following information from you for each sample you send:
  • List of samples delivered

  • Sample source (human, animal etc)

  • Sample type (EDTA; citrate; heparin; buffy coat; buccal swab etc)

  • Sample volume

  • NB: Material which is known (or suspected) to be infectious must be labelled as such and Geneservice must be told about them in advance of receipt.
What is a typical turnaround time for the DNA extraction?

 We routinely complete 200 DNA extractions in a week although higher throughput extraction can be organised on request.
How will I receive the extracted DNA?
 The extracted DNA is typically buffered in 10mM Tris, pH8 although this can be flexible according to your needs. The volume of buffer used is as follows:
  • 10ml blood sample, DNA returned in 1ml buffer

  • 200ul - 2ml blood sample, DNA returned in 200ul buffer

  • 10-50ul blood sample, DNA returned in 50ul buffer

  • FTA Paper/buccal swab sample, DNA returned in 50ul buffer

We can supply the DNA in a number of formats. However, Geneservice recommends the use of 2-D bar coded tubes. This allows greater sample security. Before accepting our quotation for DNA extraction, you will need to indicate the preferred format to receive the extracted DNA.
What happens if I want Geneservice to do some genotyping following extraction?
 We strongly recommend the DNA is stored in 2-D bar coded tubes. The samples will then be diluted appropriately for genotyping and will be passed to the relevent team. You will need to ask us for a follow up proposal of work or we can bundle the genotyping into a single project price with your extraction.
Can you help me with secure storage and retrieval for my extracted DNA samples?

 Yes. Geneservice has a DNA banking storage and retrieval system that includes making a mirrored off-site store of your samples. Please contact us for more details.
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Whole Genome Amplification (WGA)

Question Answer
What method do you use for whole genome amplification?
 We use the Multiple Displacement Amplification (MDA) method. MDA produces amplified DNA with comprehensive coverage of all gene sequences and unprecedented low bias. This revolutionary method allows researchers to rescue minute quantities of DNA and gives them greater flexibility to work with smaller samples such as buccal swabs, mouthwashes and finger pricks. In addition, a quality assessment on the amplified DNA is performed to effectively predict the usability of amplified DNA for downstream uses such as microsatellite genotyping, Taqman assays, 10K, 100K and 500K SNP microarray assays all of which are provided by Geneservice.
Can you give me more details on WGA?
 Our specifications include the following:
  • PicoGreen quantitation assay on the samples prior to amplification when the samples provided are genomic DNA

  • Either, Standard: WGA in reaction volume of 100 ul and an expected yield of approximately 100 ug of amplified DNA, or, Customised: WGA with customised yield is possible.

  • Standard quality assessment of the amplified human DNA samples, i) DNA quantitation of the WGA yield by the PicoGreen assay and ii) Loci representation QC by TaqMan assay on two loci (1004 and 699). Based on these values, Geneservice will provide a usability score for each sample that will permit the customer to decide which samples will need to be eliminated in the ensuing study

  • Delivery of the amplified product and a report
How should I send my DNA samples to you?
 The DNA samples to be amplified should be shipped in 96 well PCR plates. Each plate should have a maximum of 84 DNA samples leaving all of row H empty. The DNA samples should be in TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA), however samples in 10 mM Tris-HCl (pH 7.5) or water will also be accepted. The DNA concentration should be more than 5 ng/μl in a total volume of 20ul. The plates should be tightly sealed with strip caps and the samples frozen before shipment. Wrap each plate in some type of padded material such as foam wrap or bubble wrap before packaging on dry ice. You must pay due regard to the postal regulations that are in force at the time of sending.
How should I send other samples types to you for whole genome amplification?
 Blood, buffy coat, cell culture and blood spots to be amplified should be shipped in 96 well PCR plates. Each plate should have a maximum of 84 samples leaving all of row H empty. The total volume of the samples should be 5-50 μl with a minimum of 1000 cells/5μl. The plates should be tightly sealed with strip caps and the samples frozen before shipment. Wrap each plate and dispatch as above.
I still have some questions, who can I speak to?
 Please contact us for further information. Our specialist will be more than happy to discuss your requirements in greater details and can provide you with a custom proposal of work with pricing specific to your project.

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Taqman SNP genotyping

Question Answer
How does the Geneservice SNP genotyping contract research work?
 Geneservice need you to provide the SNP information for your project; either the surrounding sequence information with identified base change, or, specific Applied Biosystems SNP assay as detailed below. Geneservice will obtain designed assays and test them for functionality. Validated assays will be genotyped on your DNA samples to provide data in an excel format.
What technology do you use for SNP genotyping?
 Geneservice uses the 5' nuclease assay chemistry with TaqMan® MGB probes for single-tube convenience and reliable performance. Data is collected on an ABI PRISM® 7900 HT Sequence Detection System.
How many SNPs can you genotype for me?
 Geneservice can provide genotyping for any number of SNP assays. Pricing structure will reflect the improved throughput seen with larger numbers of SNP assays and samples.
How many DNAs?
 The nature of the 5' nuclease assay means that larger numbers of samples (ideally a minimum of 96) are required to enable clustering and scoring of data points. Typical project sizes are 750 to 3,000 samples for each assay. Liquid handling restraints mean that throughputs are optimised if DNA samples are provided as multiples of 384 with allowances for controls. Samples provided in this format will give the best value for money pricing.
What is your maximum throughput?

 Our current capacity is in excess of 38,000 scored genotypes a day, dependent on numbers of samples per SNP assay.
What equipment do you use to obtain such a high throughput of genotyping?
 Matrix PlateMate liquid handling robots capable of preparing in excess of 100 x 384 plates a day with low volume 5ul reactions; heat sealer; Duncan thermal cycler capable of amplifying ~150 x 384 plates in 2hrs; ABI7900 HT capable of reading 84 x 384 well plates in 3hours. Custom LIMS for sample and data tracking.
How does the 5´ nuclease assay work?
 In the 5' nuclease assay (also known as the TaqMan assay), allelic discrimination is based on the characteristic 5' to 3' exonuclease activity of Taq DNA polymerase. PCR using flanking primers is performed including fluorescent oligonucleotide probes in a homogeneous assay. The probes consist of a 5' reporter dye and a 3' quencher dye, and are specific to the region containing the base change in the region to be amplified. The 5' nuclease activity cleaves the probe if hybridisation occurs, releasing the reporter from the quencher. Two different probes with different fluorogenic reporters are put in the reaction for allele discrimination, one specific to complement each of the variant alleles to be typed. If there is a mismatch between the probe and target DNA sequence, the hybridisation is significantly reduced, therefore stopping the cleavage of reporter from quencher, and release of fluorescent signal. The amounts of each signal released indicate which allele(s) of the target region is present. The probes have been improved further by the introduction of non fluorescent quenchers, containing a minor groove binder in the probe, thereby increasing the specificity of the probe to the SNP region and increasing reaction standardisation and sensitivity.



Figure 3: Data from 5' nuclease assay reactions analysed on the ABI PRISM 7900HT Sequence detection System. The clusters of output of the fluorescent data are seen: only FAM signal, homozygous allele 1; only VIC signal, homozygous allele 2; increase in both FAM and VIC signal, heterozygous, both alleles present. The negative control is also shown.
How are SNP assays designed?
 There are three methods of designing SNP assays for use on the ABI 7900HT:
  • Assay On Demand (AOD) - These assays, currently numbering over 200,000, are pre-validated assays available from ABI. We have used ~400 of these assays in the past and have found them to be of extremely high quality.

  • Assay By Design (ABD) - These assays are designed by ABI from the provided sequence information. These assays undergo a preliminary QC by ABI, and as a result only those assays passing QC will be billed. Under normal circumstances this is our first choice for designing new assays.

  • Manual Design - These assays are manually designed by Geneservice following a strict set of guidelines. We recommend that this mode of design is used only for assays which fail ABD.


    		•
    Can you type all SNPs including deletions/insertions?
    		 All bi-allelic SNPs can be typed using this platform, including insertions/deletions; some more complicated assays eg (TT/GG) may require manual design.
    How do I supply the SNP info for the ABD and Manual design?
    		 Information regarding SNPs should be supplied to us in an Excel or tab delimited text file in the following electronic format:

    SNP nameBase changeSequence
    Turnip SNP 1A/C......AGCTTA(A/C)GTAGCT......
    Turnip SNP 2T/C......GCATA(T/C)GTCTNG......

    The sequence should contain at least 150bp either side of the SNP. The SNP of interest should be placed within brackets eg (A/C). Any additional SNPs or uncertain bases within the sequence should also be highlighted as an N. Any additional information, for example TSC / RS numbers can also be included, but are not essential.
    And what about specifying Assay On Demand SNPs?
    		 Assay On Demand (AOD) SNPs need their specific assay ID. The assay ID as shown on the ABI web site should be supplied (eg C_2608211_1) Assays can be searched for using a number of criteria from the ABI online catalogue.
    Can you design successful assays using the 5´ nuclease assay for every SNP?
    		 Although we are able to design assays for virtually any SNP - we cannot guarantee their success. The success rate depends in part on the accuracy of the sequence information given. It is therefore essential that additional SNPs in the region of interest are highlighted. It should be expected that a few assays will fail ABIs ABD design criteria / QC, although in some instances this can be overcome through manual design. We expect that about 90% of bespoke assays (ABD and manual design) will be typable.
    How do I specify my Sample information?
    		 Sample information should be supplied in an Excel file, or tab delimited text file in the following format:

    Plate nameWell referenceSample name
    Plate 1A1Turnip 142
    Plate 1A2Turnip 143
    What are the sample requirements?
    		 DNA should be ~10ng/ul, with variation amongst the sample set limited as much as possible. We require 2ul of DNA for each reaction plus an additional 30ul excess to allow for robotic overages. Excess DNA can be returned at project completion. DNA samples should be supplied in 96 well format, as these plates will be used directly on our liquid handling robots it is suggested that you use one of the following plates:
    • Skirted Abgene thermo-fast plates (Cat#AB-0800) for volumes <150ul

    • Matrix 1ml blocks (Cat#4211)

    • Matrix 2D bar-coded track mate blocks (Cat#3711) for volumes >150ul

    However we may be able to accommodate alternative skirted plates.

    A single Non-Template Control (NTC) is required on each 96 well plate, this should be in the same well position - we suggest A1. We recommend duplicating a number of samples - for example the first /last column of each 96 well plate. This enables a measure of concordance to be generated, which will subsequently increase the confidence in the results generated.
    How should I send the samples?

    		 Samples should be sent on dry ice. Please refrain from sending samples on a Thursday or Friday.
    Can you extract my DNAs if I have blood samples?

    		 Yes. Please contact us or go here
    What QCs do you carry out?
    		 We test the quality of the assay and the quality of your DNA before starting to genotype in earnest. Manually designed assays are examined using a panel of 96 control DNAs: 47 duplicated DNAs and 2 negative controls. This enables us to measure the separation, clustering and reproducibility of the assay. Assays only pass this stage if there is no ambiguity in scoring and 100% reproducibility. It should be noted that rare SNPs can sometimes be difficult to QC because there are few observations of the rare allele. Your DNA will be tested using an assay that has previously been seen to function well, poor performance can highlight the need to improve your DNAs - especially important prior to starting a large project.
    What if my assay doesn't work?
    		 We will always tell you if your assays don't work. Assays that fail ABIs design criteria / QC can be manually re-designed, in some cases this assay may also fail our QC, these can be manually re-designed using the opposite strand (where possible).
    What if my DNA doesn't work?
    		 We will always tell you if your DNA doesn't work. If your samples fail our QC it may be possible to rectify the problem through additional dilution of the samples. We have found that in some cases it is not poor quality DNA that causes the problem, but high levels of impurities. However dilution of samples can also lead to increased scatter of the plots reducing the amount of data generated.
    What impurities can impact on the quality of data?
    BSAwill not inhibit Amplitaq
    Ca2+3.5mM or greater
    CHELEX resinresin will inhibit PCR - let resin settle before transferring extracted DNA sample
    Chloroform50mM or greater
    Dimethylformamide50mM or greater
    DMSOwill not inhibit in low concentrations (1%)
    DMSOgreater than 10% will inhibit Amplitaq
    DTT1mM or greater
    EDTA50mM or greater
    Ferric ion10uM or greater
    Haemoglobin/hemeheme from porphorins will interfere with PCR
    NaCl50mM or greater
    NH4will not inhibit Amplitaq
    NP40will not inhibit Amplitaq
    Phenol50mM or greater
    KCl50mM or greater
    Propidium iodidewill not inhibit Amplitaq
    SDS50mM or greater
    Siliconized tubeswill inhibit Amplitaq
    Spermidinewill not inhibit Amplitaq
    Triton X-100will inhibit Amplitaq
    How long will I have to wait to receive my data?
    		 It always takes between 1-2 weeks to receive the assays from ABI. Production genotyping will take about a week for small numbers of samples and SNPs (eg 384 DNAs x 10 SNPs); larger projects of hundreds of SNPs and hundreds or thousands of samples may take a several months. The start date for a project will depend on current schedule and receipt of assay.
    How do you track my samples and data?
    		 Geneservice have an in-house developed laboratory information management system (LIMS) and a similarly developed SNP/primer tracking system.
    What do I do next? / I still have questions
    		 Contact us with your project details and timelines and we will prepare a proposal. Let us know how you would like to be contacted. Alternatively, we could arrange a visit or meeting.

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    Affymetrix 10k, 100k and 500k Genotyping

    Question Answer
    Tell me about these three products
    		 The Affymetrix GeneChip Human Mapping SNP arrays are a fast, economic and powerful SNP-based genetic mapping tool.

    The 10K single array product provides data on over 11,000 SNPs distributed across the genome. The 100K set is comprised of a set of two 50K arrays each with 50,000 SNPs and together, they provide data on over 100,000 SNPs across the genome. The 500K Array set is comprised of two 250K arrays, each capable of genotyping on average 250,000 SNPs. Please see below for a snapshot comparison of the 10K, 100K and 500K SNP coverage. In addition, you also have the flexibility to choose to use single 50K or 250K arrays.


    Figure 4: A snapshot comparison of 10k, 100k, 500k SNP.

    We can offer contract research with all Affymetrix SNP genotyping products.
    What are the advantages of using SNP array?
  • The SNP density enables highly powered whole genome studies.

  • It allows you to stay in charge of your budgets. A complete data set is generated from each DNA sample. This gives you the flexibility to select those samples that will be the most informative in your sample set. You can type them first, analyse your genome wide data and then decide if you need to do more.

  • The assay uses just 250ng of genomic DNA per chip - only as much as is used up on just 50 microsatellites.

  • Geneservice has set up a high-throughput mode of operation with multiple samples processed in parallel. Typically, you will get data about 2 weeks after we start processing.


    		•
    How do I get started? Using our experience and expertise from years of RNA expression analysis on the Affymetrix platform, Geneservice will process your DNA samples efficiently, accurately and economically (of course, we cannot guarantee that every project will give a significant LOD score: this depends on mode of inheritance, family structure and map location). We have kept costs as low as possible with prices starting at about £400 per sample (inclusive of chip, consumables and processing costs). Discounts may be given for large projects. We offer free advice regarding projects and data analysis. Contact us now to confirm pricing and to get a custom proposal of work.
    How should I supply my DNA? The precise amount of DNA required for each sample will be a minimum of 10ul at 50 ng /ul using reduced EDTA TE buffer (0.1 mM EDTA, 10mM Tris HCl, pH8.0). Each array requires 250ng of DNA that has preferably been tested by you for its integrity. If this is not possible please send OD260/280 ratios of samples to confirm ratio >1.8 for each sample. Samples may be accepted of a lower quality at the discretion of Geneservice but risk a higher chance of poor quality PCR.

    Sample IDs should be provided in Excel format. Samples to be tested should be sent in a 96-well format on dry ice.

    Contact us / telephone us to confirm your despatch arrangements for samples. We strongly advise that samples are not posted on a Thursday or Friday. Please post your samples to the following address:

    AffySNP,
    Geneservice/Source BioScience
    Units 24 and 25
    William James House
    Cowley Road
    Cambridge CB4 0WU
    United Kingdom
    What if my samples don't pass the post PCR quality control stage?
    		 Samples that fail the PCR QC stage will be withdrawn from the process at a cost of £100 per sample to cover consumable costs to this point, unless instructed to continue at customers risk. We would also offer customers the option of performing whole genome amplification to "rescue" their samples. It is a very successful method to obtain enough good quality DNA for further downstream processing (more info).
    When can you start my project? We will only order chips and schedule your project on acceptance of your personal quotation and receipt of the following details:
    • a signed copy of agreed quotation
    • a signed copy of terms and conditions
    • your details including purchase order with VAT exemption if it applies.
    We can only order chips and schedule your project on receipt of these details.
    When will I have to pay for the project?
    		 For large projects you will be billed at the start of the project for the Affymetrix GeneChip Mapping arrays since they need to be ordered well in advance. You will be invoiced for the balance upon completion of the project and delivery of results. You will be notified in your proposal whether up-front payment for GeneChips is required.
    How will I receive the data?
    		 Geneservice will supply you with the data from the samples on CD in strict confidence using Affymetrix software in Excel format. For example:

    SNP IDTSC IDdbSNP RS IDChrChr Location (deCODE map)pp1 control 10xba 220503Call 1Call 2Zone
    SNP_A 1513509tsc 111rs 07130556161960064173.2BB0.05
    SNP_A 1513556tsc 112rs 07130566161960122173.2AA0.53
    SNP_A 1518411tsc 256rs 094945974228867164.7AB0.08
    SNP_A 1511066tsc 864rs 0713298106725451483.4AA0.21
    This all looks great but I still would rather do microsatellites

    		 We continue to provide contract research using microsatellites and have successfully completed more than 30 genome scans using microsatellites.
    I still have some questions, who can I speak to?
    		 If you are interested in using the Affymetrix GeneChip Human Mapping Arrays or have any queries please contact us with full details of your study including the number of samples. We will return a customised project proposal, and we can schedule your work as soon as possible. Scheduling will only be arranged on receipt of a valid purchase order number.

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    Microsatellite Genotyping

    Question Answer
    What are microsatellites? Microsatellites are di-, tri-, or tetra nucleotide tandem repeats in DNA sequences. The number of repeats is variable in populations of DNA and within the alleles of an individual. The sequence below has a 20 dinucleotide repeat (40bp) stretch of CA that is shown in bold.

    CGTTCAATAAGCAAAAATCCATAGTTTTAGGAATGTGGGCT
    GCTTGGTGTGATGTAGAAGGCGCCAATGCATCTCGACGTAT
    GCGTATACGGGTTACCCCCTTTGCAATCAGTGCACACACAC
    ACACACACACACACACACACACACACACACA    GTGCCAAGCA
    AAAATAACGCCAAGCAGAACGAAGACGTTCTCGAGAACACC
    AGAAGTTCGTGCTGTCGGGGCATGCGGCGAGTAAAGGGGAT

    If you flank a microsatellite with fluorescent PCR primers then amplification will give a pair of fluorescent allelic products which will vary in size according to their repeat length. A population might possess 5 alleles which vary in size like this.

    Figure 5:

    What is genetic linkage? Genetic linkage is the tendency of two allelic features to be inherited together as an intact unit. Linkage is based upon an analysis of families. When features are close together on a chromosome there is a higher likelihood that they will be inherited together and familial recombination events will not have interfered. The likelihood that features will be inherited together is measured as a log of odds ratio (LOD) e.g. 100:1 or a LOD of 2. The statistical robustness of measuring linkage depends on testing many families so that one can really determine whether features are inherited together because they are linked or whether they are inherited together by chance alone.
    Err... you said "recombination event", whats that? A genetic recombination event is a crossover between the alleles of one of your parents. Chromosomes occur in pairs and in each pair; one will have come from your Mother and one from your Dad. If we consider a pair of chromosomes from our parents as being represented by the following, where capital letters represent the chromosome that Dad got from his Dad, and lower case from his Mum, and numbers represent the chromosome Mum got from her Dad and roman numerals the chromosomes she got from her Mum.

    Child 1 gets two un-recombined chromosomes, but child 2 inherits a recombination event on both chromosomes.

    Dad
    A a
    B b
    C c
    D d
    E e
    F f
    G g
    H h
    I I
    J j
    Mum
    1 i
    2 ii
    3 iii
    4 iv
    5 v
    6 vi
    7 vii
    8 viii
    9 ix
    10 x
    Child 1
    A i
    B ii
    C iii
    D iv
    E v
    F vi
    G vii
    H viii
    I ix
    J x
    Child 2
    a 1
    b 2
    c 3
    D 4
    E 5
    F 6
    G vii
    H viii
    I ix
    J x
    So what's linkage disequilibrium? When two features occur together as an intact unit within a population we say the features are in linkage disequilibrium. Features that occur predictably and randomly are in linkage equilibrium. Typically features in linkage disequilibrium are much closer together than those that are linked within families because in every generation since the features first occurred together there will have been an opportunity for them to become unlinked by genetic recombination thus spoiling the subsequent linkage disequilibrium.
    So how can microsatellites be used in genetic studies? Microsatellites can be used as markers in genetic studies of linkage in families and linkage disequilibrium studies of populations. In linkage studies one can examine large number of families and see when the alleles of specific markers are inherited together with a phenotype in more cases than not. Microsatellite repeat are amplified with fluorescently labeled primers and then the alleles from each individual in a family are seperated by size and the marker tested for linkage with another. The raw family data looks like this:

    Figure 6: Raw genotyping data.


    Markers in a genome screen must be no more distant from the underlying genetic cause of the phenotype than the detectable level of marker - marker linkage. This is generally about 10Mbp and so 300 or more microsatellites are required to screen the entire genome for a disease gene using linkage. Linkage is a crude tool and refining the position of a disease gene is impossible since linkage will be quickly maximized.

    In studies of linkage disequilibrium (LD), a marker allele is associated with a phenotype across an entire population. The spacing of the markers has to represent the level of LD across the genome (or locus) of the population. However, LD is not a regular phenomenon and it can extend from a few KB to many hundreds of KB. Therefore deciding where to place markers is problematic and a genome-wide screen of LD might require 500,000 markers or more to be typed per DNA. Therefore, genome-wide LD scans are not often performed and LD is more often reserved for analyzing just a few loci following a positive genome-wide linkage scan.
    Why use microsatellites rathers than SNPs? SNPs are great genetic markers but because of their low heterozygosity (the likelihood that a marker in any individual will appear heterozygous) you need to type lots of them. Microsatellites are good markers for studies of genetic linkage because they have a high heterozygosity. They are highly mutable markers with often 15 or more alleles in any given population. This means that allelic identity-by-descent can be readily established (unlike with bi-allelic SNPs) and linkage determined. However, the mutability of microsatellites can also prove problematic. Occasionally alleles can be seen to mutate within a generation giving rise to apparently non - Mendelian inheritance. This mutability becomes more of a problem when considering allelic associations within populations and it has been argued that SNPs offer a better chance of identifying marker-marker or marker-phenotype LD.
    What about study design and power? The maximum power to detect a genetic locus using linkage is restricted by the family material that one chooses to type. Linkage is refined by familial recombination and this occurs randomly. Statistical simulations of linkage studies can be made in order to determine the maximum outcome of these things and in turn whether to proceed experimentally with projects. Geneservice can help you with these analyses before you commit to genotyping. Contact us us for further information.
    Tell me about genome scans using microsatellites and linkage? A genome scan is a series of genome-wide microsatellite genotypes across a set of families. In the first instance enough markers should be typed to cover marker-maker linkage in a step-wise manner across the entire genome. Historically, we have done this by typing an established set of 400 markers that we purchase from Applied Biosystems (LMS2.5 10cM) although a denser set of markers is also available (LMS2.5 5cM). For the majority of genome screens these markers will show marker-maker linkage, however there may be loci where random recombination has restricted linkage and further markers are required. Following this genetic linkage analysis of the phenotype-genotype can begin and LOD scores established.
    How much DNA do you need per genome scan or per reaction? DNA should be supplied at a standard concentration and in a microtitre plate. We need about 5ug of each DNA for a genome scan although we might require more or less depending on how simple we find the amplification. Customers always have the option of having unused material returned to them when their experiments are completed.
    How long does this all take? Microsatellite genotyping projects can take months or just a few days. The big projects get scheduled into a vacant spot in our calendar and small projects are often combined together so that we continue to offer the best value for money to all our customers. In both cases there can be a wait of a few weeks before your experiments begin. The sooner we get things scheduled the better.
    What about after the genome scan? Data is delivered in an agreed format by CD-ROM. The post-laboratory work is in two phases, some customers want us to do both, others prefer to do all this work themselves. The first phase of work is making the genotyping calls from the raw data; the second phase is the statistical genetic analysis of the results. The agreed price will reflect the level of analysis required.
    Will you make custom microsatellite genotyping? Some customers come to us after making a genome screen and ask for a set of fine mapping microsatellite markers. However, if the established information content is already near maximum then it might be detrimental to type further markers and perhaps impossible to obtain a legitimately higher LOD score. Fine mapping markers can be used to determine allelic associations or to confirm other genetic observations. However, one should always remember that the more markers typed, the more likely an apparent statistical significance will be found purely by chance alone.
    What instrumentation have you got? We use Matrix PlateMate plus for high throughput liquid handling and a Kbiosystems Super Duncan thermal cycler. We use an ABI Prism 3100 genetic analyzer for product analysis but for very high-throughput analysis we have the option of using ABI Prism 3730 genetic analysers. All work is tracked using our in-house LIMS system.
    How much does a genotype cost? A good figure to use in your estimations is £1 to £2 per genotype including genotype calls but not including genetic analysis. This figure is not a universal quotation; rather it is just a good place to start. Genotyping sometimes works out more expensive than this but more often it is cheaper for a genome scan. Please contact us with the precise details of your project and we will provide a written custom proposal.
    How do I go about doing this research at Geneservice? This is not a something into which you should enter lightly. Geneservice will not accept liability for the costs of genotyping projects that are subsequently shown to be scientifically weak even if you have consulted us during the design of your project. We assume that most genome scans are funded from a peer reviewed grant application and as such will have had expert consideration. After receiving your funding and when considering outsourcing this work to Geneservice we encourage potential collaborators to visit us and discuss the requirements of their project directly so that our proposal can be tailored to your exact requirement. Contact us to arrange an appointment. When your project begins you will have direct access to a project manager that will manage your project at Geneservice.
    I've still got questions, wo can I speak to?

    		 OK, contact us now.

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    DNA Sequencing

    Question Answer
    How do I go about using the Geneservice DNA sequencing? These are the minimum things you need to do:
    • get your sequencing template prepared by PCR or plasmid extraction.
    • work out what primers you want to use for your sequencing; are they in our list of standard primers we supply for free?

    • fill out an on-line sequence order form including the declarations on Genetically Modified Organisms (GMO) and VAT.

    • send all of the above to the address on the bottom of the order form.
    What concentrations of DNA and primers do I need to send? We find that sequencing reactions are quite robust but we do need to know roughly what these values are. We aim for the concentrations of DNA to be 1ng/μl per 100bp for PCR products, 100ng/μl for plasmids and 3.2pmol/μl for oligos. Volumes supplied should be 6μl per sequencing reaction requested of plasmid or PCR product and 10μl of your oligos per reaction requested.
    Do PCR products need to be cleaned before being sent for sequencing? PCR templates for sequencing must have been cleaned up to remove unincorporated reactants before sending to us. In our hands all the commercial kits work OK. We will not accept liability for failed sequences when in our opinion the templates are poorly purified.
    Will you make DNA template? Yes. We have a standard protocol for extracting plasmids and for making PCRs. If you send us cultures for plasmid extraction then you must complete the declaration on GMO on the sequencing order form. If you fail to complete this then any material you send us will probably be destroyed. PCR primers must accompany PCR reaction requests. We make only one attempt at these using our standard conditions or conditions that you supply. If you decide that we need to work further on the PCR (titration) then further costs will be incurred. It is always best to contact us in advance of making template preparation requests.
    Do you make oligos? No. We are not an oligo supplier although we do have stocks of standard primers that we use at no charge to customers on their experiments. We do have a preferred oligo supplier and can get oligos synthesized quickly and cheaply for customers.
    What are your standard sequencing primers? M13F, M13R, T7F, T7R, T3, SP6, SK, KS and growing every day! The sequences are available on our web site. In addition, if you commit to 250 reads with any single oligo we will synthesize it for free and add it to the list for other customers to use. We will not disclose the origin of any oligo sequence we publish.
    Tell me about your registration to handle genetically modified organisms (GMO)? We are registered with the Health and Safety Executive (HSE) for the safe handling of your GMO. This registration is only valid if the GMO declaration is completed by you when sending cultures. We are unable to accept GMO when this declaration is not completed and your materials will probably be destroyed.
    How much does all this cost? Not very much! Sequencing prices are falling all the time and we pass on these savings immediately to our customers. However, like all businesses we recognize the benefits of economies of scale. This means that the more sequencing you send (or commit to in any year) the lower your price will be.
    So you're doing it on the cheap? On the contrary. Our sequencing operation uses over £½ million worth of hardware, including the latest ABI 3730 DNA sequencing technology.
    What experience have you got then? Geneservice has been around, in the form of HGMP, MRC geneservice and the Linkage Hotel at the HGMP-RC for almost 10 years. The sequencing team has more than 10 years experience of making reads. In the past 5 years members of our team have contributed over 2 million reads to the UK Mouse Sequencing Consortium and then the Fugu sequence finishing project.
    So you only deal with large projects? No. The minimum number of reads that we accept is 1.
    What read lengths do you get? Unlike some sequencing operations we don't make any promises on sequencing read length - we know that in isolation these promises are always wrong. On average we get about 750bp to 850bp of quality clipped sequence, although our reads are much longer at over 1kb.
    But your competitors promise longer reads than that! We can't make these promises and are surprised when others do. There are lots of reasons why sequencing reactions fail and most of them are nothing to do with us or you - that's biology. Repetitive elements and secondary structure account for short reads as will poorly purified templates. Of course a PCR product of 600bp will never give you a 650bp sequencing reaction. All of these things bring our averages down from the actual standard read length of a sequencing pass that is much higher.
    What can you do about these biological sequencing fails? We have a number of strategies to improve templates (Template Rescue Service, TRS) and to cover repetitive sequence or sequence with secondary structure (Secondary Structure Solution, SSS). We purchase some commercial reagents and have a few secret tricks for crossing these regions. Please contact us if you have a special requirement.
    Do I pay for sequencing reaction fails? Yes. We have controls in place such that we are confident that sequencing fails are generally the responsibility of the customer because of poorly prepared or purified templates, or bad oligos. You will always be sent failed read chromatograms for you to inspect them. However, when reads are sent to you, you can always ask for failed reads to be repeated and we are very happy to do so. Experience shows ~95% of failed reads fail again. In this case we will charge double for the fail if we repeat it. Obviously, if the failed read passes (5% of occasions) on repeat, we concede this to be our responsibility and you only pay once.
    What is your turn-around time? Between 1 and 2 days but this depends on the level of contract research requested and the number of reads. If you want to send us more than 90 reads at once we suggest you contact us in advance. Machine breakdown has caused turnaround time to be longer than this but we always keep a capillary sequencer in reserve.
    How is data returned to me? Most customers want their reads to be delivered by email attachment. For larger orders we can set up secure FTP sites or make CD-ROMs. Please contact us if you have any special requirement.
    How do I know you wont make an error with my important samples? We have laboratory information management systems (LIMS) in place. Your material is assigned a 96 well plate names and position when we receive it. From then on it is handled in this format and tracked with bar coding at every stage. We do not promise that mistakes cannot be made - we do promise that we have minimised these chances and that we will strive to always get you the best possible data. We will always be honest with you about your sequencing and your plates controls are always stored and are available for you to see. In the event of any dispute between customers and ourselves we will generally offer to make repeat samples for free.
    I still have some questions... OK, contact us now.

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    Quantitative / Real Time PCR

    Question Answer
    How does the rtPCR contract research work?
    		 We discuss your specific requirements, after which we would need you to provide:
    • Assay information for your project; either with the sequence for the area, or, specific Applied Biosystems assays.

    • Enough total RNA to complete your work – this will be QC'd and converted into cDNA prior to running your assays on them to your specifications.
    Results will be supplied in Excel in a mutually agreed format.
    What QCs are carried out on the samples?
    		 RNA samples are quality assessed using UV readings and the Agilent Bioanalyser. This gives an indication of the quality, and the amount of material present. Ensuring that material being converted into cDNA is of an acceptable quality.
    What technology do you use for rtPCR?
    		 We use the 5' Endonuclease assay (Taqman) to carry out real time reactions. This is a probe based technology where the assay is normally designed over an Exon / Exon boundary, this increases the specificity of the assay compared with sybr green based assays.

    What is an endogenous control / Housekeeping gene - and do I need one?
    		 When carrying out an rtPCR expression study it is very important to standardise data for variations in the extraction / conversion and running of the samples. The way this carried out is through the use of an endogenous or housekeeping gene – this will be a gene that has a fixed expression level that is unaffected by the experiment you are running. Without this control your data could be meaningless. Many researchers choose to run several control genes as an additional insurance.
    Commonly used human control genes are:
    • Eukaryotic 18S rRNA

    • Human ACTB (beta actin)

    • Human B2M (beta-2-microglobulin)

    • Human GAPD (GAPDH)

    • Human GUSB (beta glucuronidase)

    • Human HPRT1 (HGPRT)

    • Human IPC (internal positive control)

    • Human PGK1 (phosphoglycerate kinase 1)

    • Human PPIA (cyclophilin A)

    • Human RPLP0 (large ribosomal protein)

    • Human TBP (TATA-box binding protein)

    • Human TFRC (CD71) (transferrin receptor)
    How many replicate readings should I run?
    		 We would recommend that you use three replicates for each sample. Duplicate readings are the bare minimum however occasionally larger deviations are seen – especially where very low expression levels are seen, triplicate readings makes it much more likely that usable data will be obtained throughout.
    What size reaction volume do you use?
    		 The reaction volume directly affects the cost of carrying out the work; we have found that 10ul reaction volumes generally give the most cost effective way of getting reproducible data. Lower reaction volumes can be run if requested, however we have found that results tend to have greater standard deviations.
    Would the ABI Low-Density Array (LDA) be suitable for my project?
    		 The Low-density array is a recent development from ABI in which assays from ABI’s pre-validated list are spotted and dried onto a 384 array. From 11 to 380 assays can be arrayed in a limited number of arrangements. Each LDA has 8 ports each feeding 48 wells, into which your cDNA and master mix are added, once added the array is treated as a 384well plate and run on the ABI7900. The system benefits from low reaction volumes (1ul) and in our experience gives excellent reproducibility. However the main benefit is that it makes experiments with low numbers of samples, and larger numbers of assays more cost effective.
    What equipment do you use?
    		 Reactions are set up either manually using pipettes, or for larger projects a Matrix PlateMate Plus liquid handling robot is used. Plates are then heat sealed, as we have found that this greatly reduces the risk of evaporation and the associated reduction in data quality. The plates are run using an ABI 7900HT machine, and data analysed using ABI's SDS software.
    What are the sample requirements?
    		 Good quality total RNA should be supplied frozen and on dry ice. An electronic sample sheet should also be provided. Plates or tubes need to be clearly labelled. The amount of total RNA required is obviously dependent on the specific project, but for most projects 1.5-2?g of total RNA would be needed. RNA should be supplied in DEPC treated water at a minimum concentration of 100ng/ul.
    How do I specify the assays I’m interested in running?
    		 Assays can be specified in two ways:
  • 1: From ABI’s list of over 600000 pre-validated assays from human, mouse, rat, Arabidopsis, and Drosophila genes. Assays can be searched for using ABI’s search tool
    Please specify the assays using the ABI name, eg: Hs99999910_m1

  • 2: If an assay cannot found on ABI’s list then we can get assays designed using the sequence of your target area. For this we need an assay name, and the sequence with the Exon / Exon boundary central and clearly marked. Ideally with at least 150bp on either side. From this sequence an assay will be designed with a primer in each exon and a probe spanning the Exon / Exon boundary.

    		•
    How long will I have to wait to receive my data?
    		 This obviously depends on the size of the project, and our current commitments. Generally however assays take in the region of 2 weeks to be delivered and the work is normally started soon after the assays arrive, most projects take 1-2 weeks to complete the wet work and return the data. An estimate of the project completion date will be given at the start of the work.
    I still have some questions, who can I speak to?
    		 Please contact us for further information. Our specialist will be more than happy to discuss your requirements in greater detail and can provide you with a custom proposal of work with pricing specific to your project.

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    Gene Expression Microarray

    Question Answer
    How do I get started?
    		 Using our experience and expertise from over 4 years of RNA expression analysis on the Affymetrix platform, and 2 years on the Applied Biosystems 1700 system, Geneservice will process your RNA samples efficiently, accurately and economically. For the Affymetrix platform we offer 3 levels of entry giving you the flexibility to process your samples as much or as little in your own lab. You can provide us with total RNA, labelled cRNA or fragmented labelled cRNA. We offer free advice regarding projects and data analysis. Contact us now to confirm pricing and to get a custom proposal of work.
    I have a proposal – what do I do next?
    		 You should nominate a Project Leader to be our point of contact regarding your project and contact our microarrays to inform us when you would like to send samples. Please send samples Monday – Wednesday only and include your signed proposal, copy of your purchase order and completed sample annotation form for either Affymetrix microarrays and ABI 1700 Microarrays.
    How much sample should I send?
    		 This is dependant on which entry level you require:

    AFFYMETRIX
    If sending total RNA:
  • For one-cycle processing please send total RNA at a concentration of >50ng/µl in a volume not less than 13µl of DEPC-treated water.

  • For two-cycle processing please send total RNA at a concentration of >4ng/µl in a volume not less than 6µl of DEPC-treated water.

    • If sending labelled cRNA:
  • Please send 25µg of purified cRNA at a minimum concentration of 0.7 µg/µl. A sample of the original total RNA (0.1-1µg) that we will use to assess sample integrity.

    • If sending fragmented labelled cRNA:
  • Please send the fragmentation reaction to contain exactly 20µg of fragmented cRNA.


    • APPLIED BIOSYSTEMS
    If sending total RNA:
  • Please send total RNA at a concentration of >120ng/ul in a volume not less than 13ul of DEPC-treated water.


    • To maximise success you should endeavour to supply good quality samples with an OD 260/280 ratio of >1.9
    What is good quality RNA?
    		 The nature of the assay means good quality RNA is essential. At Geneservice we pride ourselves on producing good quality data and have a stringent QC system in place. We ask that all samples have an OD 260/280 ratio of >1.9 and test the quality using the Agilent 2100 bioanalyser system. Intact total RNA is represented by two strong ribosomal peaks with a 28S/18S ratio close to 2.0. Below is a profile of what is considered a good quality RNA sample:


    An example of a degraded RNA sample can be seen below. Notice the elevated baseline and decrease in the 28S/18S ratio:


    If sending cRNA we expect the profile below. A good cRNA reaction is represented by a smear of products falling between 50 and 3000 bases long.

    Further information on Quality Procedures for Gene Expression Microarrays.

    What happens if samples fail?
    		At geneservice we appreciate the importance of performing thorough Quality Control steps to ensure you obtain the best data possible. However, occasionally samples fail to produce enough labelled product due to undetectable contaminants. Under such circumstances you may wish to repeat using a fresh sample and would be charged for the processing of the initial part of the experiment again. If failure is due to our processing error, repeats would be conducted free of charge.

    How will I receive my data?
    		Data will be posted onto our ftp site into a private directory which is username and password protected. You will be supplied with this username and password by one of our project scientists which will allow you to download all data onto your personal computer on the same day the microarrays are scanned!
    Can I compare data from 2 separate projects?

    		As long as the same labelling protocol has been used throughout your projects, all data can be compared.
    I want to analyse the data myself – how do I get the probe information?

    		 We will supply all the probe information in the form of a text file for the Applied Biosystems arrays. For Affymetrix all probe information can be found through NetAffx
    What's the difference between Affymetrix and Applied Biosystems 1700? Which system should I use?
    		 The main differences between the two systems are highlighted in the table below. We believe the systems compliment each other, however both provide good, reliable data amd it mostly comes down to individual preference. Please note Applied Biosystems currently offer arrays for Human, Mouse and Rat only. For the full list of Affymetrix arrays available please click here.

    Affymetrix U133 Plus 2Applied Biosystems Human Genome Survey Microarray V2.0
    Well established systemNew system
    47,000 transcripts = 38,500 well characterised human genes29,098 genes (from public database as well as the manually curated Celera database)
    Splice variantsOver 8000 genes not covered by any other commercial arrays
    FluorescenceChemiluminescence
    25-mer probes - multiple probes per transcript60-mer probes – 87% of genes have one probe
    11 pairs of oligo probes to measure level of transcription of each sequence – multiple independent measurements for each transcript. Probes synthesized in situ on the arrayHighly annotated oracle database. Integration with PANTHER for classification of molecular functions & biological processes.
    Integrated workflow with Taqman


    Do you offer any experimental design or data analysis support?
    		 Yes, our scientific consulting service includes a free initial consultation on experiment design and various levels of expression data analysis service.
    Please go here for more information.
    I still have some questions, who can I speak to?
    		 Please contact us for further information. Our specialist will be more than happy to discuss your requirements in greater detail and can provide you with a custom proposal of work with pricing specific to your project.

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