Eukaryotic metabarcoding (EUKB01)
This course will be delivered by Dr Owen Wangensteen and will take place in Glasgow from 23rd - 27th July 2018.
Metabarcoding techniques are a set of novel genetic tools for assessing biodiversity of natural communities. Their potential applications include (but are not limited to) accurate water quality, soil diversity assessment, trophic analyses of digestive contents, early detection of non-indigenous species, studies of global ecological patterns and biomonitoring of anthropogenic impacts. This course will give an overview of metabarcoding procedures with an emphasis on practical problem-solving and hands-on work using analysis pipelines on real datasets. After completing the course, students should be in a position to (1) understand the potential and capabilities of metabarcoding, (2) run complete analyses of metabarcoding pipelines and obtain diversity inventories and ecologically interpretable data from raw next-generation sequence data and (3) design their own metabarcoding projects, using bespoke primer sets and custom reference databases. All course materials (including copies of presentations, practical exercises, data files, and example scripts prepared by the instructing team) will be provided electronically to participants.
Sunday 22nd – Meet at 43 Cook Street, Glasgow G5 8JN at approx. 17:00 onwards
Monday 23rd – Classes from 09:00 to 17:00
Session 1. Introduction to metabarcoding procedures. The metabarcoding pipeline.
In this session students will be introduced to the key concepts of metabarcoding and the different next-generation sequencing platforms currently available for implementing this technology. The kind of results that we may obtain from metabarcoding projects is explained using examples from real life. We will outline the different steps of a typical metabarcoding pipeline and introduce some key concepts. In this session, we will check that the computing infrastructure for the rest of the course is in place and all the needed software is installed. Core concepts introduced: next-generation sequencer, multiplexing, NGS library, metabarcoding pipeline, metabarcoding marker, clustering algorithms, molecular operational taxonomic unit (MOTU), taxonomic assignment.
Session 2. Metabarcoding markers. Primer design. PCR and library preparation protocols.
In this session students will learn about the various kinds of molecular markers that can be used for metabarcoding different kinds of samples and the quality of the information which can be retrieved from them. They will learn about the most commonly used primer sets for each target taxonomic group and how to use the software available for designing their own custom metabarcoding primers. They will know about sample tags, library tags, adapter sequences, PCR protocols and library preparation procedures. Core concepts introduced: metabarcoding marker, universality, specificity, taxonomic range, taxonomic resolution, primer bias, amplification errors, sequencing errors, in silico PCR, sample tags, library tags, adapter sequences, PCR, library preparation kits, PCR-free methods, avoiding contaminations, good laboratory practice.
Tuesday 24th – Classes from 09:00 to 17:00
Session 3. The OBITools pipeline. First steps and quality control.
In this session, we will start to work with the OBITools software suite, using a real sequence dataset as example for testing our metabarcoding pipeline. We will outline the steps needed to start analysing raw data from next-generation sequencers. The students will learn about the different data formats used by OBITools for working with sequences and they will perform protocols for quality control, paired-end alignment, sequence filtering, removal of chimeric sequences, sample demultiplexing, format conversion and dereplication of unique sequences. Core concepts introduced: fastq, fasta and extended fasta formats, Phred quality score, paired-end alignment, demultiplexing, sequence filtering, chimeras, dereplication, unique sequences, reads.
Session 4. Clustering algorithms. Constant and variable identity thresholds.
In this session, we will introduce different algorithms available for clustering sequences into molecular operational taxonomic units (MOTUs). We will learn the differences between constant and variable identity percent threshold for delineating the MOTUS. We will run some of these algorithms with our example dataset and will analyse the results from different methods. Core concepts introduced: MOTU, reference clustering, de novo clustering, unsupervised-learning clustering, Bayesian clustering, step-by-step aggregation methods, identity threshold, variable identity threshold, singleton sequences, sequence mapping, abundance recalculation.
Wednesday 25th – Classes from 09:00 to 17:00
Session 5. Taxonomic assignment. The ecotag algorithm. Reference databases.
In this session the students will learn about different algorithms for taxonomic assigment of MOTUs. The ecotag algorithm will be used for adding taxonomic information to the MOTUs in our example dataset and the results will be compared to those from other assignment software. Core concepts introduced: reference database, identity assignment, BLAST, phylogenetic assignment, best match, higher taxa assignment.
Session 6. Generating, improving and curating reference databases.
The quality of the reference database used for taxonomic assignment is crucial for the accuracy and applicability of the resulting datasets from any metabarcoding project. In this session the students will learn how to build local reference databases from the information available in public sequence repositories and how to add custom sequences to existing reference databases. They will also learn how sequence reference databases interact with taxonomy databases for retrieving the phylogenetic information needed by the assignment algorithms. Core concepts introduced: ecoPCR, sequence reference database, taxonomic database, taxonomic identifier (taxid), GenBank, European Nucleotide Archive (ENA), Barcode Of Life Datasystems (BOLD), SILVA database.
Thursday 26th – Classes from 09:00 to 17:00
Session 7. Refining and analysing the final dataset. Collapsing, renormalising and blank correction. α- and ß- diversity patterns.
In this session, students will learn about procedures for refining the final datasets obtained from the previous pipeline. They will learn about blank correction, renormalization procedures for deleting false positive results, and taxonomy collapsing of related MOTUs for obtaining enhanced final datasets. We will also discuss how to interpret these final datasets to obtain ecologically relevant information. Resampling and rarefying procedures are introduced. Qualitative and quantitative indices for assessing dissimilarity between samples are explained. We will introduce the UniFrac dissimilarity distance between samples, an index taking in account not only abundances of the different MOTUs but also their taxonomic affinity. Core concepts introduced: renormalization, taxonomy collapsing, blank correction, α-diversity, ß-diversity, rarefaction, MOTU richness, UniFrac distances, multidimensional scaling (MDS).
Session 8. Presenting the final results. Online resources and future developments.
In this session we will continue with the presentation of final results. Students will learn how to plot taxonomic summaries from their datasets, including krona plots, a type of graphic representation which allow to show relative abundances of reads at different taxonomic levels. The rest of the session will be dedicated to introduce current research and possible future developments of metabarcoding / metagenomics techniques and to provide a list of useful resources for further learning, continuous training and future research opportunities. Core concepts introduced: taxonomic summary, krona plots, target capture, metagenomics, mitogenomics, long range PCR, nanopore sequencing, mBRAVE.net, metabarcoding.org.
Friday 27th – Classes from 09:00 to 16:00
Session 9. Customization.
This session will be dedicated to customize individual metabarcoding projects, in function of the specific needs of the students. We will discuss the best strategies to use for obtaining good quality results from our metabarcoding projects, by optimizing time, money and computing resources. The idea is to make this session as interactive and useful as possible. We will present our current and future projects in the format of an open discussion and we will try to propose the best solutions for every potential problem in a collaborative way.
Optional free afternoon to cover previous modules, discuss data or continue with the customization session.
Email [log in to unmask] with any questions
Check out our sister sites,
www.PRstatistics.com (Ecology and Life Sciences)
www.PRinformatics.com (Bioinformatics and data science)
www.PSstatsistics.com (Behaviour and cognition)
1. June 12th - 15th 2018
SPECIES DISTRIBUTION MODELLING (DBMR01)
Myuna Bay sport and recreation, Australia, Prof. Jane Elith, Dr. Gurutzeta Guillera
2. June 25th – 29th 2018
SPECIES DISTRIBUTION/OCCUPANCY MODELLING USING R (OCCU01)
Glasgow, Scotland, Dr. Darryl McKenzie (PROTEUS)
3. July 2nd - 5th 2018
SOCIAL NETWORK ANALYSIS FOR BEHAVIOURAL SCIENTISTS USING R (SNAR01)
Glasgow, Scotland, Prof. James Curley
4. July 8th – 12th 2018
MODEL BASE MULTIVARIATE ANALYSIS OF ABUNDANCE DATA USING R (MBMV02)
Glasgow, Scotland, Prof. David Warton
5. July 23rd – 27th 2018
EUKARYOTIC METABARCODING (EUKB01)
Glasgow, Scotland, Dr. Owen Wangensteen
6. September 24th – 28th
INTRODUCTION TO MIXED (HIERARCHICAL) MODELS FOR LIFE SCIENCES USING R (IMLS01)
Glasgow, Scotland, Prof. Subhash Lele
7. October 1st – 5th
INTRODUCTION TO LINUX WORKFLOWS FOR BIOLOGISTS (IBUL03)
Glasgow, Scotland, Dr. Martin Jones
8. October 8th – 12th 2018
INTRODUCTION TO SPATIAL ANALYSIS OF ECOLOGICAL DATA USING R (ISAE01)
Glasgow, Scotland, Prof. Subhash Lele
9. October 8th – 12th 2018
INDIVIDUAL BASED MODELS USING NETLOGO AND R
Glasgow, Scotland, Dr. Andrew Jackson
Link to follow
10. October 15th – 19th 2018
APPLIED BAYESIAN MODELLING FOR ECOLOGISTS AND EPIDEMIOLOGISTS (ABME
Glasgow, Scotland, Dr. Matt Denwood, Emma Howard
11. October 29th – November 2nd 2018
INTRODCUTION TO R AND STATISTICS FOR BIOLOGISTS (IRFB02)
Glasgow, Scotland, Dr. Olivier Gauthier
Link to follow – see previous
12. October 29th – November 2nd 2018
INTRODUCTION TO BIOINFORMATICS FOR DNA AND RNA SEQUENCE ANALYSIS (IBDR01)
Glasgow, Scotland, Dr Malachi Griffith, Dr. Obi Griffith
13. November 5th – 8th 2018
PHYLOGENETIC COMPARATIVE METHODS FOR STUDYING DIVERSIFICATION AND PHENOTYPIC EVOLUTION (PCME01)
Glasgow, Scotland, Dr. Antigoni Kaliontzopoulou
14. November 19th – 23rd 2018
STRUCTUAL EQUATION MODELLING FOR ECOLOGISTS AND EVOLUTIONARY BIOLOGISTS (SEMR02)
Glasgow, Scotland, Dr. Jonathan Lefcheck
Link to follow – see previous
15. November 26th – 30th 2018
FUNCTIONAL ECOLOGY FROM ORGANISM TO ECOSYSTEM: THEORY AND COMPUTATION (FEER
Glasgow, Scotland, Dr. Francesco de Bello, Dr. Lars Götzenberger, Dr. Carlos Carmona
16. January 21st – 25th 2019
STATISTICAL MODELLING OF TIME-TO-EVENT DATA USING SURVIVAL ANALYSIS: AN INTRODUCTION FOR ANIMAL BEHAVIOURISTS, ECOLOGISTS AND EVOLUTIONARY BIOLOGISTS (TTED01)
Glasgow, Scotland, Dr. Will Hoppitt
17. 28th January – 1st February 2019
AQUATIC ACOUSTIC TELEMETRY DATA ANALYSIS AND SURVEY DESIGN
Glasgow, Scotland, VEMCO staff and affiliates
18. 4th – 8th February 2018
DESIGNING RELIABLE AND EFFICIENT EXPERIMENTS FOR SOCIAL SCIENCES (DRES01)
Glasgow, Scotland, Dr. Daniel Lakens
19. 25th February – 1st March 2019
MOVEMENT ECOLOGY (MOVE02)
Margam Discovery Centre, Wales, Dr. Luca Borger, Prof. Ronny Wilson, Dr Jonathan Potts
20. March 25th – 29th 2019
LANDSCAPE GENETIC/GENOMIC DATA ANALYSIS USING R (LNDG03)
Glasgow, Scotland, Prof. Rodney Dyer
Oliver Hooker PhD.
2018 publications -
Phenotypic and resource use partitioning amongst sympatric lacustrine brown trout, Salmo trutta. Biological Journal of the Linnean Society. DOI 10.1093/biolinnean/bly032
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