WP3 - Genotyping technology and sample handling
- Created on Saturday, 19 September 2009 15:19
- Last Updated on Saturday, 20 October 2012 12:49
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|Work package number||3||Start date or starting event: Month 1|
|Work package title||Genotyping technology and sample handling|
Description of work (WP3)
Task 3.1. Organisation of the samples
The initial task will be to collect and organise DNA samples from 25,000 cases and 25,000 controls from the different consortia that collaborate in this study: the Breast Cancer Association Consortium (BCAC), 10,000 breast cancer cases and 10,000 controls, the Ovarian Cancer Association Consortium (OCAC) 5,000 ovarian cancer cases and 5,000 controls, and the PRACTICAL consortium, 10,000 cases and 10,000 controls. These consortia have been operating for 2-3 years with excellent results. (Please see Consortium as a whole B2.3 for details). All 50,000 DNA samples will be centralised in CEGEN-CNIO where they will be quantified, normalized and organized in 96- well plate to be re-distributed to the two genotyping centres.
In order to reduce the risk of being left without DNA for some individuals, the total amount of DNA will never sent. Groups with small amounts of DNA will work with amplified DNA. This means that there will always be backups. Samples will be sent by courier to CEGEN-CNIO in 96 well-plates. The corresponding unique sample identifiers will be sent electronically and included in the CEGEN-CNIO laboratory information management system (LIMS). Plates sent to CEGEN-CNIO will be organized according to following criteria:
A majority of the third parties have already extracted DNA from blood samples. However, approximately 10,000 samples will require DNA extraction and DNA amplification. Isolation and amplification of DNA is today considered a routine and non-scientific procedure and is normally not done at research laboratories. We have chosen to subcontract this part of the WP. Please see Consortia as a whole B.2.3.
A tender process will be initiated to identify the most suitable subcontractors. We have been in contact with two potential subcontractors to learn about costs and resources. Two potential subcontractors are: (1) the Wellcome Trust Centre for Human Genetics (http://www.well.ox.ac.uk), that, if selected, will be responsible for genotyping of the prostate cancer patients (n=10,000) and controls (n=10,000) (2) The CEGEN-Barcelona (http://www.cegen.org), located at the Centre for Genomic Regulation (http://www.crg.es), that, if selected, will perform the high-throughput genotyping on the ovarian cancer samples (5,000 cases and 5,000 controls). Below we have listed the main reasons for including subcontractors.
An Illumina custom array will be prepared for genotyping using the Golden Gate technology. All SNPs selected will be checked to pass the in silico design criteria being suitable for assay development. A SNPScore for each marker will be provided by Illumina – values of >0.6 (out of 1) means that the SNP is included. A low SNPScore could be due to several reasons: assay outside limits, SNPs in duplicated and repetitive region, another SNP within 60 nucleotides away, or tri-or quad-allelic SNPs. We anticipate that this will be rare, because all SNPs will have been identified through previous genome-wide studies with similar design requirements. For those failing SNPs, we will determine whether another SNP in strong linkage disequilibrium can be used as a replacement, or whether another genotyping technology will be required for this SNP.
Plates will be sent to CEGEN-Barcelona and Wellcome Trust-Oxford by courier and unique sample identifiers will be sent electronically. As a quality control, plates with 90 Coriell samples (30 trios) will be also genotyped for each of the 1,536 SNP oligo pool assays. Plates will be included in each genotyping centre’s own LIMS in order to facilitate the control and management of samples. The Illumina standard protocol will be followed at all three genotyping centres, starting with 5 ul of DNA at a concentration of 50ng/ul. Five ul of water will be added to the plates. Following, start the Illumina protocol adding 5 ul MS1.
Once all genotypes have been generated, each genotyping centre will upload results in a repository provided by CEGEN-CNIO (SNPator). This web-based tool will be used as a centralised data repository and will save backups on a regular basis. The repository will permit genetic data to be integrated into specific databases for analyses by WP2 in order to identify the best 50 SNPs for stage III, please Task 3.3.
In addition, and in the absence of genome wide scans for modifiers of BRCA1 which might identify genes that only act epistatically with BRCA1, Georgia Chenevix-Trench, partner 3, will request funds from the National Health and Medical Research Council of Australia (NHMRC) to genotype the 3,000 BRCA1 carrier DNAs for 1,536 SNPs from genes involved in response to ionizing radiation also using the GoldenGate technology. Putative modifiers identified from this approach will be genotyped in the rest of CIMBA using the technologies described in Task 3.3.
Different genotyping platforms will therefore be used. Platforms to be used include Taqman, Sequenom iPlex technologies and Veracode. For groups using Taqman, assays will be evaluated and optimized at UCAM, partner 2, in 90 Coriell samples (trios) and distributed to each participant group. For each of the SNPs, these 90 Coriell samples will be also sent for genotyping as a control. For those groups that do not have the capacity, genotyping in stage III will be done at CEGEN-CNIO.
Platforms to be used include Taqman, Sequenom iPlex technologies and Veracode. For groups using Taqman, assays will be evaluated and optimized at the UCAM, partner 2, in 90 Coriell samples (trios) and distributed to each participant group. For each of the SNPs, these 90 Coriell samples will be also sent for genotyping as a control. Individual genotyping centres will ensure that cases and controls are mixed on 96-well plates that include at least 1 empty well. A minimum of 3% of samples will be genotyped in duplicate. Genotyping data and cluster images will be uploaded to a repository develop by WP2 for centralised quality control (QC). Essential QC information for each genotyped SNP include: concordance in Coriell samples, concordance in duplicate samples, call rate, and consistency with Hardy-Weinberg equilibrium.
Plates with less than 90% call rate will be excluded. After centralised quality control, all genotype data will be uploaded to the repository of the WP2 Consortium database For BRCA1 and BRCA2 carriers, genotyping will be carried out using Sequenom iPLEX and Veracode technologies. For 6,000 of the 15,000 available BRCA1/2 carrier DNA samples have already been centralised and plated in Brisbane, Australia, and the 100 candidate SNPs (50 ovarian + 50 breast cancer) will be genotyped there by iPLEX (funds to be requested from the Australian NHMRC).
These genotypes will be incorporated into COGS in order to increase the statistical power. For the remaining cases, we plan to use Veracode platform from Illumina that permits the study of 96 SNPs simultaneously. Results will be analysed by WP2 in order to identify the best susceptibility SNPs for each of the three diseases. In summary, the funding sought for the three steps is distributed as follows: Stage I. Genome-wide SNP scans have already been performed by several groups. Data will be sent to UCAM nine months after the start of the project. Costs for the stage I genotyping are not covered by COGS. Stage II. High-throughput genotyping will be performed by one partner and two subcontractors. Third parties will contribute DNA samples and COGS will pay for shipment of the samples. Stage III. Genotyping will be performed by third parties not included in Step I or II. COGS will provide the reagents and control samples.
|These data will be used to evaluate the association between each genotype and disease, combined over all available studies. To allow for differences in the genotype platforms used in each genome scan, we will use imputation methods to estimate genotypes at all known SNPs, using the international HapMap as a reference. We will then perform statistical tests of association for each known SNP against disease. We will also provide a web-based tool to visualise results from all the genome-scan data (and, subsequently, the follow-up results). Once the initial combined analyses are complete, we will identify a set of up to 1,536 SNPs for each disease, representing the most promising loci, for further genetic analyses as described in WP3 (task 3.2). First identifying the most significant SNPs, and then using multiple regression approaches to define a set of independently significant SNPs for further genotyping. Simultaneously, those loci with strong evidence of association will be passed to WP4 for fine-scale mapping.|
D3.1 Samples shipment to CNIO from other groups (month 9)
D3.2 Samples preparation for shipping and genotyping (month 12)
D3.3 Design and synthesis of Illumina Custom Arrays (month 14)
D3.4 Generation of genotypes (stage II) of the three diseases (50,000 samples) (month 22)
D3.5 Evaluation of assays for Taqman/iPlex/Veracode (month 28)
D3.6 Data replication and generation of genotypes (stage III) (month 33)