Worked Examples

While THAPBI PICT stands for Phytophthora ITS1 Classification Tool, with appropriate primer settings and a custom database, it can be applied to other organisms and/or barcode marker sequences.

These worked examples use public datasets from published papers, with various markers covering oomycetes, fungi, animals and plants. The main criteria has been mock communities with known species composition.

• Environmental Phytophthora ITS1 - A simple example using the default primers and database. Based on a paper from earlier in the THAPBI Phyto-Threats project:

  Riddell et al. (2019) Metabarcoding reveals a high diversity of woody host-associated Phytophthora spp. in soils at public gardens and amenity woodlands in Britain. https://doi.org/10.7717/peerj.6931

• Environmental Oomycetes ITS1 - An example where the defaults can be used, but ideally requires a different primer pair and a custom database. Based on:

  Redekar et al. (2019) Diversity of Phytophthora, Pythium, and Phytopythium species in recycled irrigation water in a container nursery. https://doi.org/10.1094/PBIOMES-10-18-0043-R

• Drained fish ponds 12S - An example with a single marker and custom database. Based on:

  Muri et al. (2020) Read counts from environmental DNA (eDNA) metabarcoding reflect fish abundance and biomass in drained ponds. https://doi.org/10.3897/mbmg.4.56959

• Fungal Mock Community ITS1 & 2 - An example with multiple markers (including two sequenced together) requiring separate primers settings and databases, based on:

  Bakker (2018) A fungal mock community control for amplicon sequencing experiments. https://doi.org/10.1111/1755-0998.12760

• Great Lakes Mock Community 16S - An example with two mitochondrial markers (sequenced separately), with mock communities, where we focus on the minimum abundance threshold. Based on:

  Klymus et al. (2017) Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes. https://doi.org/10.1371/journal.pone.0177643

• Bat Mock Community COI - A single marker example in bats, showing importance of the database content with the default classifier. Based on:

  Walker et al. (2019) A fecal sequel: Testing the limits of a genetic assay for bat species identification. https://doi.org/10.1371/journal.pone.0224969

• Synthetic controls with fungal ITS2 - A single marker example in fungi, with mock biological communities and synthetic control sequences. Based on:

  Palmer et al. (2018) Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data. https://doi.org/10.7717/peerj.4925

• Soil Nematode Mock Community - Four markers (sequenced separately) in a soil nematode mock community. Based on:

  Ahmed et al. (2019) Metabarcoding of soil nematodes: the importance of taxonomic coverage and availability of reference sequences in choosing suitable marker(s) https://doi.org/10.3897/mbmg.3.36408

• Pest Insect Mock Communities - Three markers (sequenced together) in insect mock communities. Based on:

  Batovska et al. (2021) Developing a non-destructive metabarcoding protocol for detection of pest insects in bulk trap catches https://doi.org/10.1038/s41598-021-85855-6

• Endangered Species Mixes 16S etc - A dozen markers in animals and plants (sequenced together). Based on:

  Arulandhu et al. (2017) Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples. https://doi.org/10.1093/gigascience/gix080

For each worked example there is a different sub-folder in the THAPBI PICT source code under examples/ containing at least setup.sh to do one-off setup like downloading the public data, and run.sh to execute the main analysis discussed. There will usually be assorted other files like reference sequences, or metadata.tsv.

Running the examples will create or use subdirectories raw_data/ for the downloaded FASTQ files, intermediate/ for per-sample working files, and summary/ for the final output reports. Where the example includes positive controls like mock communities, the expected species content is recorded under expected/ in per-sample files.