This course is being delivered by Dr. Owen S. Wangensteen, an expert in the application of molecular techniques to the study of marine ecological issues and biodiversity assessment of marine benthic ecosystems, including high-throughput sequencing and bioinformatics. He is currently working at
the University of Salford (UK), actively participating on the development of new metabarcoding techniques for the assessment of biodiversity in the marine realm (both in benthic and seawater samples), as a member of Project SeaDNA (NERC, UK). The course will run from Monday 20th to
Friday 24th February 2017 in Berlin, Germany.
Overview:
Metabarcoding techniques are a set of novel genetic tools for
qualitatively and quantitatively 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, diagnosis of health status of fisheries, early
detection of non-indigenous species, studies of global ecological
patterns and biomonitoring of anthropogenic impacts. This workshop gives
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 workshop, 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 at 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. This
workshop will review mostly techniques and software useful for eukaryotic
metabarcoding. Another workshop focused on procedures currently used in
microbial metabarcoding will be available from Physalia-courses.
Teaching format:
The workshop is delivered over ten half-day sessions (see the detailed
curriculum below). Each session consists of roughly a one hour lecture
followed by two hours of practical exercises, with breaks at the
organizer’s discretion.
Assumed background:
No programming or scripting experience is necessary, but some previous
expertise using the Linux console and/or R will be most welcome. All
examples will be run in a Linux environment. Thus, either a Linux PC
or a virtual box running Linux under Windows or Mac environment will
be needed. MacOSX systems might be OK, although installation of some
additional Python packages might be needed in that case. The syllabus
has been planned for people which have some previous experience running
simple commands in Linux and using the R environment (preferently RStudio)
for performing basic plots and statistical procedures. You will need
to have a laptop with Python 2.7 installed for running OBITools, the
main metabarcoding software package we will be using during the course,
but no experience with Python is necessary. If in doubt, take a look at
the detailed session content below or contact Dr. Owen S. Wangensteen
(owenwangensteen@gmail.com mailto:owenwangensteen@gmail.
Course programme:
Monday 20th – 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. The kind of
results that we may obtain from metabarcoding projects is explained
using examples from real life. I will outline the different steps of a
typical metabarcoding pipeline which will be further reviewed along the
course. I 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: 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 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 know about the most commonly used primer sets for each target
taxonomic group and they will learn to use 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.
Tuesday 21st – Classes from 09:30 to 17:30
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. Fixed 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 methods with fixed
and variable identity percent threshold for delineating the MOTUS. We
will run some of these algorithms with our example dataset and will
analyse the differences in the results from the different methods. Core
concepts introduced: MOTU, reference clustering, de novo clustering,
unsupervised-learning clustering, Bayesian clustering, multi-step
aggregation methods, identity threshold, variable identity threshold,
singleton sequences, abundance recalculation.
Wednesday 22nd – Classes from 09:30 to 17:30
Session 5. Taxonomic assignment. The ecotag algorithm. Reference
databases.
In this session the students will learn about different algorithms for
taxonomic assignment 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, assignment of higher taxa.
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 for the assignment algorithms. Core
concepts introduced: ecoPCR and ecoPCR format, sequence reference
database, taxonomic database, taxonomic identifier (taxid), GenBank,
European Nucleotide Archive (ENA), Barcode Of Life Datasystems (BOLD),
SILVA database.
Thursday 23rd – Classes from 09:30 to 17:30
Session 7. Refining the final dataset. Collapsing, renormalizing and
blank correction.
In this session, students will learn about procedures for refining the
final datasets obtained from the previous pipeline. They will learn about
blank correction, renormalizing procedures for avoiding false positive
results due to cross-sample contamination, taxonomy collapsing of related
MOTUs and other algorithms for obtaining enhanced final datasets. Core
concepts introduced: cross-sample contamination, renormalization,
taxonomy collapsing, blank correction.
Session 8. Analysing the final dataset. α- and ß- diversity patterns.
We will discuss how to analyse and interpret the final datasets
resulting from our metabarcoding pipelines, so to obtain ecologically
interpretable information. Resampling and rarefying procedures for
taking in consideration the different number of total reads of each
sample (sampling sizes) are introduced. Measures of α-diversity and
qualitative and quantitative indices for assessing the dissimilarity
between samples (ß-diversity) are explained. We will also 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: α-diversity, ß-diversity,
rarefaction, MOTU richness, UniFrac distances, multidimensional scaling
(MDS).
Friday 24th – Classes from 09:30 to 17:30
Session 9. 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.
Session 10.
Optional free afternoon to cover previous modules and discuss data.
Application deadline is the 20th of January 2017.
