D: Building OTU phylogeny

Note

In this section we will talk only about OTUs, but if you wanted to work at the ASV level you could simply use ASV sequences in place of OTU sequences. So whenever you see OTU in this section, you can simply replace this with ASV for equivalent results.

Introduction

One of the major benefits of metabarcoding over traditional methods of elucidating ecological community structure is that as part of identifying taxonomic units, we automatically acquire DNA sequences for each of our taxonomic units. This allows us to use these OTU sequences to construct phylogenetic trees for our meta-community, paving the way for analyses that go beyond just community composition into phylodiversity and phylogeography. As well as providing more detailed information about the relationships among our OTUs, phylogeny can help to better understand what our OTUs are in terms of taxonomy and evolutionary placement, potentially informing us about traits and other evolutionary properties.

Data

The starting point for this section is a FASTA file of sequences from which you want to build a phylogeny, i.e. ASVs or OTUs. If you worked through the previous sections, these data were produced during filtering or OTU delimitation. You wil also need some reference data, as described in the relevant tutorials.

You can download a zip archive of example data for all of the tutorials in this section here. Unzip this to a convenient location. If you’re working through the tutorials using the example data from previous sections, this zip archive contains all the reference data you will need for the relevant tutorials.

Building a phylogeny

In this section, we will look at two ways to build a phylogeny with your OTUs. We would like to stress here that the focus is on exploring the possibilities and potentialities of phylogenetic metabarcoding, but a detailed exploration of phylogenetic methodologies and analysis is beyond the scope of these resources. The broad methodologies presented here cover a general outline of methods for building phylogenies, but the specific alignment and tree-building software employed has been chosen for speed and ease of use, rather than necessarily for optimal phylogenetic reconstruction.

First, we will look at direct phylogenetic reconstruction based solely on your OTU sequences in the Making a barcode tree tutorial.

Second, we will look at how you can add your OTUs to an existing phylogeny based on full mitochondrial genomes to greatly improve accuracy of your resulting tree in the Phylogenetic placement tutorial.

The third section takes a small detour to work through a pipeline for building a mitochondrial genome tree from freely available data for cases where you don’t necessarily have this data already to hand. See the Building a mitogenome Tree tutorial.

Next steps

We suggest you start with the Making a barcode tree tutorials and follow the tutorials in order, although each is standalone and does not require the outputs from a previous step.

If you have your own data, we suggest nonetheless following these tutorials with the available example data first, as it is small enough to rapidly produce outputs and get you familiar with the steps. Then, you may wish to build your own mitogenome tree with reference data suitable for your project (following Building a mitogenome tree) then place your OTUs on this tree (following Phylogenetic placement).