Development of optimal wet-lab and bioinformatics protocols for implementation of RAD sequencing for NERC science

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

RAD ABSTRACT A common problem in ecological genetics is the development and deployment of markers in wild populations. This can be a time-consuming and expensive task for non-model organisms, and can be a serious block to achieving research goals. Recently, restriction-site associated DNA sequencing (RADSeq) has emerged as a technology with the potential to simultaneously discover, validate and score robustly a large number of markers (in the thousands) across any genome. Using ultra-high throughput sequencing technologies it is possible to screen hundreds of individuals per week. Two issues hinder wide take-up of this technology: the difficulties encountered in preparing samples for RADSeq, and the analysis of the tens to hundreds of millions of sequence data points generated. Here we propose to establish in the GenePool genomics facility (a collaborating centre in NERC's NBAF) the resources and know-how to deliver this game-changing technique to UK environmental and population genetics research. We will develop best-practice, streamlined and repeatable laboratory methods that will deliver -robust methods for generating RADSeq libraries from large sample sizes, -optimisation of the application of molecular indexing (to permit multiplexing), -proof of the use of multiple different restriction enzymes (sampling independent populations of sites) and -systems for robust paired-end sequencing (to scan longer regions per RAD site for SNPs). There are no validated software tools for analysis of RADSeq data, and our exploration of the small datasets we have developed in house suggest that patterns of error in sequences and differential representation of sites in datasets makes data processing non-trivial. We will build easy-to use pipelines for RADSeq data analysis, incorporating best-practice quality checking, error management and outputs ready for further analyses in third party software. These pipelines will be used to verify the mapping of dauer entry and other traits in the C. elegans model system, and to deliver genetic analysis of RAD sites in the other genomes. These tools and protocols will subsequently be offered in-house to NERC science, and also disseminated through training and publication. We will also make available the validated RADSeq adapter sets at cost to NERC science. We will use three test systems. The major testbed will be a set of recombinant inbred lines derived from, and newly constructed crosses between, wild strains of the nematode Caenorhabditis elegans, where we will investigate the use of RADSeq markers in fine mapping of traits in a fully-sequenced genome. We will also construct test libraries from two other organisms, the oak Quercus robur and the burying beetle Nicrophorus vespilloides, to examine RADSeq in larger genomes, and in organisms with no genome data existing.

Publications

10 25 50
 
Title Transmissions exhibition 
Description The Blaxter lab collaborated closely wth artists-in-residence (see http://www.ascus.org.uk/ciie-micro-residency-artists-announced/) in the Centre for immunity Infection and Evolution to inspire and be part of the final exhibition "Transmissions". Mark Blaxter appears in the film work produced by Anne Milne, and the work of the lab inspired Jo hodges and Robbie Coleman to produce a piece dedicated to the lab. 
Type Of Art Artwork 
Year Produced 2014 
Impact 'Transmissions' was showcased to the general public within a group exhibition 'Parallel Perspectives' in Summerhall as part of the Edinburgh International Science Festival 2015 art programme, How The Light Gets In . This exhibition of work susequently travelled LifeSpace, Dundee, returning to Edinburgh to showcase at the Tent Gallery, Edinburgh College of Art. 
URL http://www.ascus.org.uk/ciie-micro-residency-2/
 
Description RAD-Seq is an exciting technology that allows us to look at the genetics of all sorts of traits in wild animals and plants. Using RAD-seq we can start to understand the biology and diversity of many, many more species globally.
Exploitation Route RAD-Seq is alsready being used across environmental science to investigate may aspects of biology.
Sectors Agriculture, Food and Drink,Environment

 
Description We have established a community website to promote, explain and coordinate efforts in RAD sequencing in the UK. RADSeq is now widely used to develop genetic resources for crop organisms and endangered species, assisting in directed breeding programmes.
First Year Of Impact 2011
Sector Agriculture, Food and Drink,Environment
Impact Types Economic

 
Description BBSRC Project Grant (RAD Sequencing)
Amount £71,505 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2010 
End 11/2012
 
Description BBSRC Project Grant (Ruff)
Amount £176,281 (GBP)
Funding ID Bb/J018791/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2013 
End 09/2015
 
Description BBSRC project grant (Striga)
Amount £43,647 (GBP)
Funding ID BB/J011703/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2015
 
Description BBSRC/DFID Project Grant (Ash Dieback)
Amount £162,746 (GBP)
Funding ID NORNEX 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 04/2015
 
Description Daphne Jackson Trust Fellowship
Amount £80,457 (GBP)
Organisation University of Surrey 
Department Daphne Jackson Trust
Sector Charity/Non Profit
Country United Kingdom
Start 08/2011 
End 08/2014
 
Description TSB (Atlantic salmon SNP chip)
Amount £323,669 (GBP)
Funding ID 100965 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 07/2011 
End 07/2014
 
Description NERC NBAF 
Organisation Natural Environment Research Council
Country United Kingdom 
Sector Public 
PI Contribution We are a funded node of the NERC Biomolecular Analysis Facility. This collaboration involves three other University facilities.
Collaborator Contribution We are charged with delivering research support, sequencing and bioinformatics to NERC funded researchers.
Impact The collaboration is multi-disciplinary, involving genetics, genomics, bioinformatics, nmetabolomics and transcriptomics. The outputs include dozens of research papers and several collaborative grants.
Start Year 2006
 
Description Blaxter group presentations and outreach 2015 
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 Mark Blaxter and research team communication and outreach 2015

Globodera genomics and blobtools software
25/02/2015 JHI Postgraduate Student Competition 2015 James Hutton Institute, Aberdeen, UK A tale of Two Peaks: Analysing Genomic Data from Potato Cyst Nematodes Talk
26/03/2015 JHI Cell and Molecular Sciences (CMS) seminar James Hutton Institute, Invergowrie, Dundee, UK Frustration and happiness : (De)-constructing parasite genomes Talk
16/06/2015 JHI Dundee effector consortium (DEC) meeting 2015 Birnam Arts and Conference Centre, Birnam, UK Variation within the Globodera pallida species complex: preliminary results Talk
03/09/2015 Molecular and Cellular Biology of Helminth Parasites IX Bratsera Hotel, Hydra, Greece Inter- and intra-specific analyses of the effector complement in potato cyst nematodes Poster
18/09/2015 UoE Postgraduate Poster Day University of Edinburgh, Edinburgh, UK Inter- and intra-specific analyses of the effector complement in potato cyst nematodes Poster
26/09/2015 Edinburgh University Doors Open Day University of Edinburgh, Edinburgh, UK Potato Cyst Nematodes (PCN) - Nematode parasites of potatoes Poster
30/11/2015 NextGenBug University of Edinburgh, Edinburgh, UK Blobtools: Blobology 2.0 Talk
01/12/2015 UK pollinator genomics meeting Roslin Institute, Edinburgh, UK Bees and Blobs Talk

LepBase
06/03/2015 EMARES Cambridge, UK The Bicyclus Genome Project Talk
06/03/2015 EMARES Cambridge, UK An introduction to Lepbase Talk
17/06/2015 Arthropod Genomics Manhattan, Kansas, USA Lepbase - A multi genome database for the Lepidoptera Poster
24/07/2015 10th Heliconius Meeting Gamboa, Panama Lepbase - A multi genome database for the Lepidoptera (API demonstration) Workshop
24/07/2015 10th Heliconius Meeting Panama Lepbase - A multi genome database for the Lepidoptera Poster
26/07/2015 10th Heliconius Meeting Panama Lepbase Workshop Talk
04/09/2015 Edinburgh Bioinformatics Edinburgh, UK Lepbase - A multi genome database for the Lepidoptera Talk
26/09/2015 Open Doors Day "Make a butterfly" interactive exhibition
26/09/2015 Edinburgh University Doors Open Day Edinburgh, UK Lepbase Multiple Sequence Alignments game Poster+Game
28/10/2015 NextgenBUG Dundee, UK Lepbase - an Ensembl (and more) for the Lepidoptera Talk

Nematode genomics
24.06.2015 20th International C. elegans Meeting Los Angeles USA A new evolutionary framework for the phylum Nematoda: a case study of HOX cluster evolution Poster
24.06.2015 20th International C. elegans Meeting Los Angeles USA Caenorhabditis Genomes Project Workshop (organiser and chair) Talk
24.06.2015 20th International C. elegans Meeting Los Angeles USA Current status of the CGP in Edinburgh Talk

Meloidogyne genomics
10-14 August 2015 ESEB Lausanne-Switzerland Genomic consequences of hybridization and the loss of meiotic recombination in Root-knot nematodes poster
15-18 December 2015 PopGroup Edinburgh-UK Genomic consequences of hybridization and the loss of meiotic recombination in Root-knot nematodes talk
23 February 2016 NextGenBug Edinburgh-UK Genomics of Root-knot nematodes talk
Year(s) Of Engagement Activity 2015
 
Description Press releases and website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We have engaged actively with the University press office to promote press coverage of our research outcomes, particularly major publications (which have had coverage in national and international newspapers) and in blogs and other online media. We have also promoted major new initiatives such as additional core funding of the Edinburgh genomics facility.

Increased visibility of Edinburgh Genomics within the community; requests for comment by funders and government on matters pertaining to genomics.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016