Welcome to the To the Canopy and Beyond repository!

This repository is a collection of several scripts and mini-tutorials guiding you through the methods of metabarcoding analyses which were performed in the paper by Jauss & Nowack et al., 2021. The data and methods are based on the paper by Jauss & Walden et al., 2020 and the corresponding Github repository FromForestSoilToCanopy.

Most scripts here deal with the data for oomycetes, but they were also applied for Cercozoa if not stated otherwise. The raw data can be downloaded here, plots and figures were generated with the final OTU tables (not provided) and annotation files accessible in the folder 00_Data.

Relevant scripts and tutorials can be found in the corresponding markdown files, which are linked and highlighted for each category.

This folder contains the final OTU table, taxonomic annotation, sample metadata as well as the oligosheet for demultiplexing the raw data. Intermediate files are not provided here, but you can generate them yourself by following the next steps.

In this pipeline, you find the neccessary scripts to generate the (unfiltered) OTU table from raw .fastq files.

This is a collection of several scripts neccessary for the taxonomic annotation of our OTUs. What we do first is to BLAST against the NCBI nt database to remove contaminants. After that, we download and process public oomycete/cercozoan sequences, which we use as a reference database for our taxonomic annotation with vsearch.

The visualisation of the taxonomy then includes a diagram showing the total taxonomic composition. What's more, we also visualised the Taxonomy per sampling season in a sankey diagram

This section provides scripts on how to import, explore and filter the OTU table with Qiime, how to extract sequences from the filtered table and last but not least how to paste the filtered metadata into the filtered table.

Here we deal with the methods of how to plot alpha diversity indices in a boxplot including pairwise significance testing. Also, we performed rarefaction analyses to test if a higher sequencing depth would have revealed more OTUs.

One of the most straightforward methods of visualising beta diversity is an NMDS plot, the script is provided here. But we also plot the number of shared OTUs in a Venn diagram.