Establishing Single Molecule Real Time Sequencing for the North of the UK

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

Over the last 5 years molecular biology has been transformed by the application of new sequencing technologies which allow DNA and RNA to be sequenced much faster than was previously possible and at a significantly reduced cost. We are now at the cusp of a second transformative wave of technologies with the availability of third generation sequencing methodologies that enable single molecule, real-time sequencing. These developments create opportunities for researchers working within the BBSRC remit.
The proposal seeks to place a Pacific Biosciences RS II high-throughput sequencing platform within the Centre for Genomic Research (CGR) at the University of Liverpool, a facility that has provided DNA sequencing and analysis services to the UK research community for over 5 years. The CGR is embedded within the Technology Directorate (TD) at the University of Liverpool (http://www.liv.ac.uk/technology-directorate/), to ensure utilisation of assets by the Faculty and thus drive academic excellence. In considering the financial sustainability of an SRF, the TD considers a number of less tangible returns, such as publications, student training and grant proposals as important components of the Faculty's core business, alongside returns such as access fees. There is hence a strong commitment by the University of Liverpool to genomics as an enabling technology. As such, the CGR is one of the largest facilities of its type in the UK, with a core staff of 20 people. The CGR handles 200 - 300 projects per year, ranging from small pump-priming / R&D projects to large-scale projects involving the sequencing and analysis of several hundred samples. To make best use of the capacity and expertise residing in the CGR and the wide skill sets of laboratory and bioinformatics staff, the proposal Investigators collaborate with researchers across the academic spectrum to assist them in devising, executing and analysing genomic datasets. The CGR currently offers access to a range of high-throughput sequencing platforms: Roche 454FLX+, Illumina HiSeq and MiSeq as well as the Ion Torrent platforms. Our partner in this bid, Edinburgh Genomics, formed from the fusion of GenePool and Roslin's ARK-Genomics, operates in a very comparable manner and extensive links already existing between the two facilities and applicants on this proposal.
We now request a 46% contribution to the purchase of a Pacific Biosciences RS II to extend the current range of platforms. The PacBio system is the only available platform on the market that will offer amplification-free sample preparation in combination with the potential for very long read output. Hence, the technology is ideally suited to the application in areas of core strategic fit with BBSRC priorities, namely Food Security, Bioenergy and Industrial Biotechnology as well as Basic Biosciences underpinning Health. The majority of research within the laboratories of the applicants is funded by the BBSRC. We have identified five key applications of the technology such as the structure and regulation of complex genomes [(eg wheat) or the diamondback moth (Plutella xylostella), one of the most important pests of cruciferous crops], understanding haplotypes in livestock as well as investigating the composition of microbial communities as applicable to health and biotechnology.
In addition to underpinning research at the University of Liverpool, the applicants have also received widespread support from across Northern Universities and Scotland, highlighting further projects that will benefit from the application of the technology. Industrial partners (Oxitec, an SME, and Unilever) have pledged support, as has the University of Liverpool. As a consequence, this is a highly cost effective and relevant investment that will place an instrument with high user demand in an environment with a proven track record of delivering high quality science with skilled staff and a clear access model for academia and industry.

Technical Summary

The Centre for Genomic Research (CGR) at the University of Liverpool and Edinburgh Genomics have provided DNA sequencing and analysis services to the UK research community for over 5 years. The CGR is one of the largest facilities of its type in the UK, with a core staff of 20 people. As a facility, the CGR handles 200 - 300 projects per year, ranging from small pump-priming / R&D projects to large scale projects. The current sequencing infrastructure within the CGR comprises Ion Torrent PGM, Roche 454 and Illumina platforms. As such, the CGR offers access to high-throughput sequencing technologies and applications thereof to a large number of projects that are aligned with the major strategic priorities of the BBSRC (e.g. pest control, crop and livestock genomics/transcriptomics, metagenetics/metagenomics).
This proposal seeks to expand capabilities through the purchase of a Pacific Biosciences RS II platform, the only third generation sequencing platform currently on the market. The PacBio RS II offers parallel, uninterrupted observation of natural DNA synthesis by a single DNA polymerase, allowing data to be captured quickly without amplification bias. The last two years have seen real improvements to the technology and a re-assessment of the strengths of the platform. We therefore believe that the time is opportune for the CGR to apply the technology to projects within the BBSRC's remit.
To achieve this, we have brought together a group of Investigators from Liverpool and Edinburgh with a strong track-record of BBSRC funding. In addition, and in order to demonstrate the breadth of science and critical mass of users, we have received extensive support from collaborating groups across the N8 Northern and Scottish Universities and Industry and now request a 46% contribution to the purchase of a PacBio RS II system. Lastly, we will promote uptake by academic and industrial partners though marketing, training/workshop activities and a pump-priming competition.

Planned Impact

We propose explicit outreach and training activities to bring information on the capabilities and capacity available through the Centre for Genomic Research to potential users and other stakeholders. The Investigators' key strengths are in genome sequencing, genomic technology and bioinformatics and we aim to make an impact from our research through:
1. Assisting collaborators in pump-priming work, in designing experiments, in applying for funding and in publishing their results, depositing data in public repositories, and exploiting commercial potential of findings. Researchers working with the CGR and Edinburgh Genomics will get the benefit of having trained staff handle their project and having access to cutting edge equipment. We will therefore have an impact on many different disciplines in basic and applied sciences and on the research councils and charities that fund them.
2. Working with The University of Liverpool's Business Gateway to identify potential areas for commercial exploitation of the work handled though the CGR. We also have embedded a project/business manager within the CGR to drive this area forward.
2. Reaching out to the research community and the general public through formal and informal presentations, marketing material and social media. In this, we will utilise the effective press communications systems within the University of Liverpool to communicate our work to wider audiences, and use public forums such as open days, social media and our website to further publicise our work. The CGR is also part of a wider UK community of academic service laboratories with whom we co-organise the annual UK Next Gen Meeting, to ensure the relevance and reach of what the CGR offers as a facility.
3. Training of researchers in the applications of next generation sequencing through targeted training events in collaboration with our partners in Scotland with whom we share the post of an MRC funded training bioinformatician. There is a recognized skills gap in next generation sequencing training provision. The CGR is committed to running at least three annual training courses and - in collaboration with Edinburgh Genomics - will run Master classes and residential summer schools as of mid 2015, coordinated and delivered by a dedicated training bioinformatician: (1) Master classes, aimed at researchers of all seniorities and delivering an overview of the technologies, their applications, the core algorithmic underpinnings of next-generation genomics analysis, as well as hands-on experience in using some of the analytical tools. (2) Residential Summer Schools, focused on reaching the coming generation of researchers: PhD students, postdoctoral researchers and early career fellows and lecturers. They will include hands on experience of next generation sequencing, in depth training in the key analytical technologies, and a programme of lectures on applications from guest lecturers and specialists. The aim is for these events to become self-funding by 2015.
4. Encouraging all CGR staff and staff associated with research grants led by the Investigators to be actively involved in these activities. In addition, the CGR is active in training PhD students and postdocs. There are 6 PhD students working within the CGR and we run numerous courses and give presentations on our science internally and externally.
5. Working closely with the technology platform providers to drive innovation and test new methodologies and applications. The CGR has a close working relationship with Roche, Illumina and Agilent, acting as a reference site and certified service provider for applications. We will also continue to host manufacturer-led seminars as events aimed at providing networking opportunities between academics and commercial partners.
6. Benefiting industry by providing a complete service for contract sequencing and bioinformatics. This raises the competitiveness of UK companies and therefore helps employment within the UK.
 
Description We have purchased a Pacific Biosciences Single molecule sequencer and have made it available to academic and industry scientists. We have held training days and a symposium to inform scientists
Exploitation Route This facility will be of value for scienctists in microbiology. cancer research, comparative genomics, HLA typing etc.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

URL http://www.liv.ac.uk/genomic-research/
 
Description The equipment used award has been used in various industrial studies that has resulted in economic benefit to the partners
Sector Agriculture, Food and Drink,Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms. 
Description Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences' Single Molecule, Real-Time (PacBio SMRT) sequencing technology. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Method used in BBSRC funded DNA foundries 
 
Description Collaborations with Unilever 
Organisation Unilever
Department Unilever UK R&D Centre Port Sunlight
Country United Kingdom 
Sector Private 
PI Contribution Genome data generation to underpin programs in personal and home care divisions of Unilever.
Collaborator Contribution Provision of materials.
Impact Better understanding within Unilever of microbial communities as relevant to personal and home care.
Start Year 2013
 
Description Materials Innovation factory 
Organisation Unilever
Department Unilever UK R&D Centre Port Sunlight
Country United Kingdom 
Sector Private 
PI Contribution We are partners in a open innovation space
Collaborator Contribution Unilever are co-locating personell and contributing to equipment and building costs
Impact none
Start Year 2012
 
Description School visits 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Numerous school visits to the CGR
Year(s) Of Engagement Activity 2015,2016