Monday – Classes from 09:30 to 17:30
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. Some examples of results that can be obtained from metabarcoding projects are explained. We will outline the different steps of a typical metabarcoding pipeline and introduce some key concepts. We will also explain the format of the course. 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: high-throughput sequencing, multiplexing, NGS library, metabarcoding pipeline, metabarcoding marker, clustering algorithms, molecular operational taxonomic unit (MOTU), taxonomic assignment.
Session 2. Molecular laboratory protocols. DNA extraction. Metabarcoding markers. Primer design. PCR and library preparation. Good laboratory practice.
In this session we will learn the basics about molecular laboratory procedures needed for metabarcoding. While there will be no hands-on laboratory practices, guidelines and best practices for all key laboratory steps will be discussed. We will explain sample collection techniques, including eDNA and bulk community samples, pretreatment and DNA extraction protocols. The diverse molecular markers available for different kinds of samples and target taxonomic groups will be discussed. The students will learn to design and test custom metabarcoding primers. They will know about sample tags, library tags, adapter sequences, PCR protocols and library preparation procedures.
Core concepts introduced: good laboratory practice, proper sample collection, bulk (community DNA) and eDNA samples, DNA preservation, DNA extraction, PCR, clean up, metabarcoding marker, universality, specificity, taxonomic range, taxonomic resolution, primer bias, amplification errors, sequencing errors, DNA contaminations, in silico PCR, library generation, sequencing platforms, sample indexing, adapter sequences.
Tuesday – Classes from 09:30 to 17:30
Sessions 3 & 4. The USEARCH pipeline.
In this session, we will work with the USEARCH and VSEARCH software suites, using a real sequence dataset as example for testing our metabarcoding pipeline. We will outline the steps needed to start analysing raw data from high-throughput sequencers. The students will learn about key bioinformatics workflows and they will perform quality control, sample demultiplexing, paired-end merging, sequence filtering, removal of chimeric sequences, format conversion, dereplication of unique sequences, sequence clustering as well as taxonomy assignment using reference databases. We will run most commands in an R environment using a user friendly modular wrapper script, with specific focus on when and why each module is necessary. We also introduce mBRAVE as an easy to use cloud based platform as well as cutting edge methods to obtain Exact sequence variants / haplotypes from metabarcoding data.
Core concepts introduced: fastq and fasta formats, Phred quality score, paired-end
alignment, demultiplexing, sequence filtering, chimeras, dereplication, unique sequences, reads, singleton sequences, abundance recalculation, OTU clustering, sequence repositories, identity
assignment, BLAST, GenBank, Barcode Of Life Datasystems (BOLD), mBRAVE, LULU, read denoising, haplotyping.
Wednesday – Classes from 09:30 to 17:30
Session 5. The OBITools pipeline I. Workflow, first steps and quality control. Clustering algorithms with variable thresholds.
In this session, we will work with the OBITools software suite, using the same dataset we used in USEARCH for testing some alternative metabarcoding pipelines from a Linux terminal environment. We will also introduce different algorithms for clustering sequences into MOTUs, such as CROP and SWARM. We will learn the differences between constant and variable identity threshold for delineating the MOTUS.
Core concepts introduced: reference clustering, de novo clustering, unsupervised-learning clustering, Bayesian clustering, step aggregation methods, hard identity threshold, flexible identity threshold.
Session 6. The OBITools pipeline II. Taxonomic assignment using ecotag.
In this session we will continue with the OBITools pipeline. We will learn about phylogenetic algorithms for taxonomic assigment. 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. The students will learn how to build local reference databases from the information available in public sequence repositories and how to add new custom sequences to these local reference databases. They will also learn how sequence databases interact with taxonomy databases for retrieving the phylogenetic information for the assignment algorithms.
Core concepts introduced: local reference database, phylogenetic assignment, best match, assignment of higher taxa, ecoPCR and ecoPCR format, taxonomic database, taxonomic identifier (taxid).
Thursday – Classes from 09:30 to 17:30
In this session, students will learn about procedures for refining and curating the final datasets obtained from the previous pipelines. 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 compare the results from the different pipelines tested and we will discuss how to interpret them in order to obtain ecologically relevant information.
Core concepts introduced: renormalization, taxonomy collapsing, blank correction.
Session 8. Presenting the final results. α- and ß- diversity patterns.
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 graphic representation showing relative abundances of reads at different taxonomic levels. Resampling and rarefaction procedures for assessing biodiversity patterns will be introduced. Qualitative and quantitative indices for assessing dissimilarity between samples will be 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 affinities.
Core concepts introduced: taxonomic summary, krona plots, α-diversity, ß-diversity, rarefaction, MOTU richness, UniFrac distances, ordination techniques, multidimensional scaling (MDS).
Friday – Classes from 09:30 to 17:30
Session 9. Experimental design. Customization.
In this session we will learn how to design a successful metabarcoding project and how to customize it in function of the specific needs. We will discuss the best strategies for obtaining good results by optimizing time, money and computing resources. The idea is to make this session as interactive and useful as possible. We will present some 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. 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 discussed: optimal multiplexing, ecological replication, technical replication, sequencing depth, price per sample.
Session 10. Hands-on project brainstorming
In small groups the participants will have the opportunity to plan and develop metabarcoding projects based on their research questions and taxonomic groups of interest. Project proposals will be presented and discussed in the form of 5-minute presentations, and they will be evaluated and improved by interacting with workshop participants. We will finish the workshop with an interactive open questions session.
Core Concepts: Experimental planning, developing successful research projects and proposals, concept evaluation and improvement by peer review, using metabarcoding as a tool to answer exciting research questions.