Introduction and rationale

The British 1958 birth cohort1 is based on all persons born in Britain during one week in March in 1958. Participants have been followed throughout their lives and biomedical information has been collected at various time points. Biological samples were collected from the cohort during medical examinations between Sept 2002 and March 2004. Funding for creation of a blood derived DNA bank was provided by the MRC (strategic project grant G0000934). Funding for creation of lymphoblastoid cell lines, cell line derived DNA extraction, banking and distribution was funded by the Wellcome Trust (grant no 068545).

DNA and cell line banks were created in the ALSPAC Laboratory. Blood derived DNA is available from 8018 individuals and has been used successfully in several genotyping studies.

Cell lines from 2288 individuals were created at ECCAC, Porton and a further 5239 in the ALSPAC laboratory. DNA has been extracted from all of these samples and has been organised into geographically representative subgroups of samples specifically for use as control samples in case control studies. Samples have been distributed to over 20 collaborators and were used as control samples by the Wellcome Trust Case Control Consortium2.

Further funding was obtained in 2006 and extended in 2007 for management of the cell line and DNA banks and distribution of samples (grant number GR079996). This funding will end on 31st May 2008 and we are therefore applying for additional funds to continue these functions.

The ALSPAC laboratory

The ALSPAC Genetic Epidemiology Laboratory is part of the Department of Social Medicine, University of Bristol. The department has a strong track record in genetic epidemiology. The department currently has custodianship of samples for the Avon Longitudinal Study of Parents and Children (ALSPAC), Caerphilly, Speedwell, Boyd Orr, Christs Hospital, Barry-Caerphilly studies.

The ALSPAC laboratory is equipped to create and manage biological sample, DNA and cell line banks and currently actively manages sample collections for ALSPAC and the 1958 birth cohort. The ALSPAC laboratory team works closely with the Bristol Genetics Epidemiology Laboratory (BGEL www.bgel.genes.org.uk) which was established by Professor Ian Day in 2004. The group also has a bioinformatics core led by Dr Tom Gaunt.

Prof George Davey-Smith is director of ALSPAC and has recently been awarded an MRC centre CAiTE - Centre for Causal Analyses in Translational Epidemiology, which opened in September 2007. The centre has recruited 5 academics working in the genetic epidemiology field hence increasing the expertise in this area within the department.

The ALSPAC, BGEL and MRC Centre laboratories and sample stores will move to a new purposed designed facility in summer 2008.

The laboratory is equipped for high throughput cell line production and DNA processing. Two custom designed robotic cell maintenance systems are used for lymphoblastod cell line growth. Two DNA processing robots (Tecan Genesis Freedom 2000, and Beckman Biomek 2000) are used to quantitate DNA samples with picogreen, normalise the concentration, and prepare plates for distribution to genotyping centres. A Quadra 96SV is also available for production of 384 well plates.

The laboratory is licensed by the Human Tissue Authority for storage of human tissue for research purposes and has developed custom designed Laboratory Information Management System to log processes and track samples. Samples are stored in secure cryostores and freezer stores. These are alarmed and a member of staff is contactable automatically 24hrs a day in the event of a freezer failure to prevent loss of samples.

The laboratory is a member of the Public Population Project in Genomics (P3G) DNA quantification project that was set up in 2006 to establish internationally recognised standards in DNA quantification. DNA samples from the laboratory have been successfully genotyped in a number of different laboratories using a variety of methods including the Affymetrix 500K human mapping array and Illumina Human Hap550 genotyping Bead Chip high throughput systems.

Genotyping

The laboratory distributes DNA to collaborators for genotyping although for simple SNP genotyping for the ALSPAC study we have established links with K Biosciences (http://www.kbioscience.co.uk/). The company hold stocks of DNA from the ALSPAC cohort and collaborators are encouraged to use the company. This is cost effective in terms of DNA use and genotyping costs and has simplified quality control processes. We propose to set up a similar arrangement for investigators using 1958BC DNA.

Receipt and storage of genotyping data and quality control The Laboratory has experience of handling genetics data.All genotyping data generated from ALSPAC study samples is returned to the laboratory and incorporated into an Oracle database. Before inclusion various quality control checks are run on the data including ensuring data is in Hardy-Weinberg Equilibrium and comparisons of genotypes from duplicated samples specifically included in sample sets for quality control purposes. Where data do not appear to be of suitable quality the problems are discussed with the genotyping laboratory and appropriate steps taken to improve the results. Documentation regarding the genotyping method and outcome of quality control checks is stored with the data.

Proposal for 1958 Sample ManagementAdministrative duties for 1958BC Interim Oversight Committee

All proposals to use 1958BC DNA need to be approved by the 1958BC Interim Oversight Committee. Dr Ring represents the Bristol laboratory on the committee and Dr Wendy McArdle will continue to advise regarding the feasibility of providing samples to users and technical advice regarding proposals.

Completion of a Material Transfer Agreement (MTA) is necessary before samples can be released to users. We understand that Professor Paul Burton's group in Leicester will undertake the administration associated with MTAs but they will continue to be signed off by the University of Bristol.

Ethical Approval

An application to extend the current ethical approval to include generic approval for genetic analysis, similar to that in place for the ALSPAC study will be coordinated by the Bristol team in order to allow the transfer of genetics data to Bristol and distribution to collaborators as approved by the Interim Oversight Committee.

DNA Bank Management

DNA has been extracted from all blood samples and cell lines although the yield from some samples was lower than others. Our current funding has covered extraction of further cell line pellets from low yield samples but only from those cell lines originally prepared in Bristol. By June 2008 there will be at least 400mg of DNA available from all such cell line samples but stocks of some ECCAC prepared cell lines are significantly lower. The maximum amount of DNA distributed from any sample in the last 12 months is 20mg. Therefore no further extractions will be necessary from the samples established in Bristol in the next 2 years assuming requests continue at a similar rate. However further regrowth and extraction of some of the 2288 samples produced at ECCAC will be required. Stocks of these cell lines were transferred to Bristol recently to enable us to restock the DNA bank when necessary.

DNA Distribution

We will continue to distribute DNA to users who have been approved by the 1958 Interim Oversight Committee. Dr Wendy McArdle will continue to liaise with users regarding supply of samples and any technical issues.

DNA is supplied to collaborators either as standard issue plates (1micro-g per well at 50ng/ micro-l) or at bespoke concentrations and volumes for large genotyping projects. In the last 12 months we have supplied 21 standard issue plates and prepared 5 bespoke requests. We anticipate that the level of requests for DNA will increase in the next 24 month period as results from genome wide association studies become available. We will continue to maintain a stock of "standard issue" plates for immediate issue from stock to collaborators and where necessary prepare samples at bespoke concentrations and volumes for large genotyping projects. Receiving laboratories will continue to be asked to cover the cost of transport. Requests for "cherry picked" samples, ie provision of a subset of samples which need to be individually picked from stock plates rather than an aliquot of all samples in a given plate can be accommodated. However, we would need to ask the requestor to cover the costs of the additional staff time involved in sample preparation as we have done for such requests in the past.

In addition we will set up an arrangement for providing a single SNP genotyping service withK Biosciences (http://www.kbioscience.co.uk/) if required. A stock of 1958BC DNA will be sent to the company and orders for genotyping placed via the Bristol laboratory in order to ensure that all genotyping has been approved by the 1958BC Interim Oversight Committee. In order to test this set up and associated quality control monitoring we have asked for funds to run 5 SNP genotypes on all samples. Details of the SNPs selected will be submitted to the oversight committee for approval before genotyping.

Please note that for some requests we require information about previous genotyping or phenotypes in order to select specific samples or plates of samples. We do not have access to this information in Bristol and are currently advised which samples are required by Professor David Strachan. In order to continue to provide samples selected on the basis of phenotype or previous genotyping results we will need to be advised by someone who has in depth knowledge of all 1958 data. We understand that the Centre for Longitudinal Studies will provide this support since the Bristol team currently have no access to phenotypic data.

Receipt of Genetics Data

The Bristol group has considerable experience of handling genetics data. If required we would set up systems similar to those used for the ALSPAC study to handle genetics data for the 1958 cohort. Quality control checks will be carried out including comparison of results from control samples and verifying that results are in Hardy Weinberg Equilibrium. Any problems highlighted by the quality control checks will be discussed with the genotyping lab and genotyping repeated or excluded if necessary. Results will be stored on a genetics results database designed specifically for the 1958 cohort study but based on the design of systems currently used in the laboratory. Data would be released in agreed formats when necessary. If required summary data can be provided via a website. Several Bristol staff currently have the expertise to oversee the development of such quality control, database and web site management but such processes are time consuming therefore we would require funding for a full time researcher to develop and manage the system and a fulltime data preparation assistant to assist with data cleaning and distribution.

Cell Line Bank Management

The cell line bank is established and aliquots of all cell lines are stored in Bristol with backups stored at ECCAC, Porton Down. Samples will continue to be kept in a viable condition in secure cryovessals. Sample will be regrown to replenish DNA stocks as described above. No costs for regrowth and provision of cell lines for other purposes, eg expression studies, have been included in this proposal but such studies could be facilitated if extra funds were made available to cover the staff and consumable costs.

The costs of back up storage at ECCAC are covered until December 2010 from previous 1958 funding therefore no further contribution to cover ECCAC costs is required for the duration of this proposal.

Storage of Biosamples

If required the Bristol laboratories would be able to store and distribute other biological samples held by the 1958BC. Costs have been included to cover storage of samples; distribution costs would need to be assessed on a case by case basis.