Eukaryotic metabarcoding (EUKB01)
Delivered by Dr. Owen Wangensteen
This 5 day course will run from 23rd - 27th July 2018 at Margam Discovery Centre, Wales.
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.
Intended Audience: This workshop is mainly aimed at researchers and technical workers with a background in ecology, biodiversity or community biology who want to use molecular tools for biodiversity research and researchers in other areas of bioinformatics who want to learn ecological applications for biodiversity-assessment. In general, it is suitable for every researcher who wants to join the growing community of metabarcoders worldwide.
Monday 20th 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 21st 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 22nd – 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 23rd 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 24th 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.
Session 10. Optional free afternoon to cover previous modules, discuss data or continue with the customization session.
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