High throughput intermediate scale sequencing for the Midlands

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

Since the advent of sequencing, driven by Frederick Sanger, the knowledge of the underlying genetic code of an organism has been a key feature in establishing the mechanisms by which inheritance influences phenotype. The rapid explosion in next generation sequencing technology in the past decade has taken us from huge consortia trying to determine the sequence of a single human genome to individuals being able to sequence their own genomes for close to $1,000. Alongside the sequencing of the genomic DNA it is also possible to sequence the transcriptome (genes expressed in a cell or tissue) or investigate modifications to the bases of DNA that alter the expression of genes. Researchers at the University of Nottingham have used NGS to investigate changes in the copy number of sequences within the genome and even used NGS to investigate how genomes are copied during bacterial replication.
Although the UoN has a range of sequencing technologies available, there is a clear gap at two levels of sequencing that are addressed by this proposal. Current technologies available at UoN allow sequencing at the level of individual microbial genomes or targeted sequencing of small regions of the human genome. These scales preclude the use of NGS to study diverse organisms with larger genomes where many of the techniques developed for studying well-characterised genomes can be readily applied. At the opposite scale, whole genome sequencing (WGS) of small genomes can be of enormous importance when carrying out both targeted and random mutagenesis experiments to engineer new phenotypes (synthetic biology). Here the generation of compensatory or off target mutants is a risk and so routine WGS can be applied to screen against such events. At this scale, the labour costs associated with library preparation limit the cost effectiveness of these approaches.
To address these issues we seek to purchase a NextSeq500 sequencer (Illumina) alongside a BioMEK 4000 liquid handling robot that will be capable of preparing 24 independent libraries for sequencing at any one time. The NextSeq technology can theoretically sequence 96 libraries in a single run enabling cost effective high throughput sequencing of multiple bacterial genomes simultaneously. The BioMEK 4000 can also be used to prepare a wide range of libraries suitable for sequencing drastically reducing overhead costs to researchers. The cross compatibility of libraries generated between all the Illumina sequencing platforms will allow DeepSeq to prepare libraries which can be sequenced using capacity at our partner institutions.
NGS technologies have a wide range of research questions. The NGS technologies outlined here will greatly enhance research at the UoN in a broad range of fields. Some of these fields include: (i) Synthetic Biology (ii) Molecular Microbiology (ii) Industrial Biotechnology and Bioenergy (iv) Food Security (v) Regenerative Medicine (vi) Stem Cell and Developmental Biology (vii) Healthy Aging (viii) Epigenetic Basis of Aging and Disease (ix) Bioscience for Health (x) Systems Biology and Bioinformatics (xi) Exploiting New Ways of Working.
The development of an enhanced NGS facility at the UoN alongside the establishment of a regional Midlands Sequencing Consortium will enable the UoN and collaborators to address key biological questions in the above areas which will have a significant impact in science, the economy and society.

Technical Summary

Our goal is to provide the University of Nottingham (UoN) with a high throughput-sequencing platform supporting vertebrate genome scale projects to massively parallel microbial genome sequencing. Two key technologies are coupled to provide a fast turnaround-sequencing platform suitable for medium scale projects. We will acquire a NextSeq 500 Illumina sequencer. This represents the latest generation of Illumina technology, using a more efficient two-dye sequencing process reducing reagent costs for a single sequencing run on the platform. The NextSeq provides rapid sequencing with an average 27hr run generating the same quantity of data as a HiSeq2500 in the same time at equivalent consumable costs. This will allow researchers at the UoN and further afield to carry out proof of principle experiments alongside small-scale sequence based analyses. We will also purchase a liquid handling robot to generate reproducible controlled libraries at high throughput providing significant savings in time and resource. This will also allow us to generate multiple libraries to analyse on a single NextSeq run enabling routine sequencing of small genomes, an approach which is vital for both Synthetic Biology and Industrial Biotechnology and Bioenergy approaches. Together this equipment will enable UoN to support the broadest possible range of NGS applications, serving all domains of life from bacteria to plants and vertebrates. The establishment of the Midlands Sequencing Consortium will extend these approaches to other Universities beyond the UoN. DeepSeqs (the UoN sequencing facility) well established track record in supporting sequencing projects within the UK and internationally will ensure the equipment achieves maximal usage. Importantly, the compatibility of technologies and approaches we are developing with the wider community ensures that projects can be readily scaled up to high-throughput approaches with the currently established sequencing centres such as TGAC.

Planned Impact

Over the last ten years the scale of sequencing achievable within individual laboratories has shifted from single plasmids to whole genomes. The establishment of next generation sequencing is key to answering many of the questions driving biology today. There are two scales of sequencing where we anticipate significant impact. Firstly massively parallel sequencing of bacterial scale genomes utilising robotic library preparation and secondly whole genome sequencing of human scale genomes. Due to the multidisciplinary research areas to which DeepSeq contributes, the main objective for the facility with this new equipment will be to have an impact on research, researchers and industry from the following disciplines:
(i) Synthetic Biology: NGS will enable the identification of novel proteins and processes with the potential for industrial and academic impact.
(ii) Molecular Microbiology: the research proposed will have a significant impact in a wide range of industrial and academic sectors where understanding the impact of individual mutations and the interplay between them.
(iii) Industrial Biotechnology and Bioenergy: sequencing rationally engineered (targeted and evolved) microbial chassis for process improvements in the manufacture of chemicals and fuels.
(iv) Food Security: NGS is essential for characterizing variation occurring within diverse populations of plants and animals.
(v) Regenerative Medicine/Stem Cell and Developmental Biolgoy: the new technology at DeepSeq will enable genome, transcriptome and DNA modifications level understanding of the development of specialized cells from stem cells and broader issues associated with vertebrate development.
(vi) Healthy Aging/Epigenetic Basis of Aging and Disease/Bioscience for Health: Numerous projects focused on Aging and Health will significantly benefit from rapid access to NGS technologies.
(vii) Systems Biology and Bioinformatics/Exploiting New Ways of Working: the impact of NGS data on systems biology is dramatic. The research proposed here will address methods to extract meaning from the volume of data generated.
DeepSeq will also have an impact on:
a. Researchers: Through the acquisition of formalized training in NGS technologies. These will include both researchers within the UoN, members of the wider Midlands Sequencing Consortium and further afield.
b. The University of Nottingham: Having NextSeq/automated robotics will attract new collaborations to Nottingham especially in areas requiring massively parallel sequencing or human genome scale sequencing.
c. The Midlands: Shared resource with compatible sequencing platforms across multiple Midlands Universities including Sheffield and Leicester alongside the M5 grouping.
d. The international relationships between Nottingham University and researchers outside the UK through collaborations between Nottingham researchers and the international research community including international companies.
The wider public will also benefit in the longer term from the research conducted at DeepSeq through the increased ability of different industrial sectors to respond to their customer needs from the environment, to agriculture and health.
The research achievements from DeepSeq and the Midlands Sequencing Consortium will be communicated to a range of audiences via presentations through to discussions and workshops with industry contacts, publications in journals targeting a wide range of audiences and conferences. The research from DeepSeq and its potential will also be communicated to the general public through the yearly University of Nottingham May Fest and through the 'Nottingham Potential' outreach activities.

Publications

10 25 50

 
Description This project was to provide higher capacity sequencing to the Midlands in the form of a Next Seq Illumina platform, robotics and associated computational resource. We also sought to establish the midlands sequencing consortium - to share our pooled resources throughout the midlands. This has been very successful with meetings occurring in Nottingham and Sheffield to date. We also have a number of developing collaborations with other universities including Birmingham and Leicester.
Exploitation Route The midlands sequencing consortium is an ongoing project and will continue to support NGS applications in the midlands and further afield.
Sectors Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description This award has led to the establishment of ongoing collaboration between industry and academia. The award in and of itself did not generate "findings" but rather served to establish a sequencing resource in the midlands and foster collaboration and active discussion between academia and industry partners.
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Advisor to UK BioBank Genomics Policy
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description A New Durable Read EXtension Method for Very, Very Long Reads
Amount £798,242 (GBP)
Funding ID 212965/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2019 
End 01/2022
 
Description From Comparative Genomics to Comparative Genetics - What is Required for Life Without DNA Replication Origins?
Amount £495,280 (GBP)
Funding ID BB/R007543/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2018 
End 07/2021
 
Description Studying potential interplay between active demethylation and WT1-dependent transcriptional regulation during glial differentiation.
Amount £502,112 (GBP)
Funding ID BB/N005759/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2019
 
Description Read Until EBI 
Organisation EMBL European Bioinformatics Institute (EMBL - EBI)
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed a website and interface for the analysis of minION data (minoTour) - we have also developed the first implementation of read until - selective sequencing on the minION sequencer.
Collaborator Contribution The EBI are world leaders in algorithm and storage development.
Impact Grant Submission to the BBSRC
Start Year 2016
 
Description Real Time Analysis 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed minoTour - a real time suite of software for analysis of minION reads.
Collaborator Contribution Edinburgh have developed poRe - an R based suite for the analysis of minION data.
Impact Grant application to the BBSRC
Start Year 2015
 
Description The Telomere-to-Telomere (T2T) consortium is an open, community-based effort to generate the first complete assembly of a human genome. 
Organisation National Institutes of Health (NIH)
Department National Human Genome Research Institute (NHGRI)
Country United States 
Sector Public 
PI Contribution I have been contributing expertise, time and sequencing data to the activities of of the telomere-to-telomere consortium. The goal of this consortium is to sequence the first human genome from telomere-to-telomere. Our expertise through the Long Read Club has been exploited to enable this goal.
Collaborator Contribution Other partners have generated sequencing data, analysed and assembled reads and presented this work.
Impact No outputs to date.
Start Year 2019
 
Description The Telomere-to-Telomere (T2T) consortium is an open, community-based effort to generate the first complete assembly of a human genome. 
Organisation University of California, Santa Cruz (UCSC)
Country United States 
Sector Academic/University 
PI Contribution I have been contributing expertise, time and sequencing data to the activities of of the telomere-to-telomere consortium. The goal of this consortium is to sequence the first human genome from telomere-to-telomere. Our expertise through the Long Read Club has been exploited to enable this goal.
Collaborator Contribution Other partners have generated sequencing data, analysed and assembled reads and presented this work.
Impact No outputs to date.
Start Year 2019
 
Description Zika 
Organisation University of Birmingham
Department Institute of Microbiology and Infection
Country United Kingdom 
Sector Academic/University 
PI Contribution We are working to develop a protocol for real time sequencing and analysis of the Zika Virus. We are providing real time sequence analysis and manipulation of the sequencer via minoTour and our read until scripts to ensure uniform depth of coverage for the zika virus in real time.
Collaborator Contribution Birmingham are leading a coordinated bid on Zika sequencing.
Impact A grant submission to the MRC
Start Year 2015
 
Title minotour v 1 
Description Minotour is a real time set of tools for analysis of nanopore data. 
Type Of Technology Software 
Year Produced 2019 
Open Source License? Yes  
Impact This is being used across a number of our projects. 
URL http://minotour.nottingham.ac.uk
 
Description Grand Challenges in Genomics - Invited Panel Speaker - Joint meeting of the NHGRI/Wellcome Trust, London, Feb 2019 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Grand Challenges in Genomics was a meeting to discuss the next ten years of Genomics and the ways in which both NHGRI and the Wellcome Trust should target investment and funding in the future.
Year(s) Of Engagement Activity 2019
 
Description Long Read Club 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Long Read Club is an informal grouping of users interested in exploring long read sequencing technologies in all their guises. We are raising awareness of methods, best practice and experience. This is being done through a website, twitter account and youtube channel. Over 900 have signed up to the email list, nearly 700 followers on twitter and over 130 people have subscribed to the youtube channel.
Year(s) Of Engagement Activity 2019
URL http://youtube.com/c/longreadclub
 
Description Singapore Genome Centre - Porecamp Singapore Training Course - Lead Instructor and Keynote - Sept (2018) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Porecamp is an instructional course for using nanopore sequencing in the lab and the field. It is open to all and serves to increase the uptake of nanopore sequencing globally.
Year(s) Of Engagement Activity 2018
 
Description University of British Columbia - Porecamp Training Course - Lead Instructor and Keynote - May (2018) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Porecamp is a training course to encourage uptake of Nanopore sequencing in the field and laboratory.
Year(s) Of Engagement Activity 2018