How to do Diet DNA Metabarcoding from Animal Faecal Samples

How to do Diet DNA Metabarcoding from Animal Faecal Samples

Description of the data and file structure

Project: How to do Diet DNA Metabarcoding from Animal Faecal Samples

Date: 30/08/2025

Authors: Rachel D. McConnell and Crinan Jarrett, Diogo F. Ferreira, Luke L. Powell, Alma L.S. Quiñones, Davide M. Dominoni, Andreanna J. Welch

Purpose: These data files may be used to run an example bioinformatics workflow and perform statistical analysis of metabarcoded Dietary DNA from birds and bats.

The data is comprised of:

1. ZBJ_Sequence_files containing dietary sequencing data from 277 samples collected from birds and bats across 16 farms in Cameroon
2. Qiime2 bioinformatic output files including the final 16-farms-ASV table
3. An NCBI COI ZBJ database and classifier
4. Statistical Analysis including the bird and bat metadata, R scripts to clean and organise the diet data, perform basic generalised linear model, a multivariate analysis and a network analysis. The plots and results produced by the scripts are also included.

Files and variables

File: Statistical_Analysis_Files.zip

Description: This folder contains the bird and bat dietary ASV Table "16Farms_ASV_Table.csv" and the
metadata files, "bird_and_bat_capturedata.csv" and "faeces_sample_database.xlsx". The folder also contains the R script used for statistical analysis. This includes "1.organize&clean_metabarcoding.R" which contains a function to filter and organise the ASV table for statistical analysis, the file "2.glm_examples" which contains code to conduct GLMs and a multivariate analysis and the "3.building_analysing_networks" file which contains code to perform a network analysis of the dietary information. Missing values are indicated as "NA".

File: NCBI_Database.zip

Description: This folder contains the COI reference sequences and taxonomy downloaded from NCBI using RESCRIPt and the output files generated from this tutorial https://forum.qiime2.org/t/… (Robeson et al., 2021; Rognes et al., 2016). The COI classifier created from the ZBJ reference sequences and taxonomy produced by Qiime2 is also included, "zbj-ncbi-classifier.qza".

File: ZBJ_Sequence_Files_1.zip

Description: This file contains dietary DNA from birds and bats sampled in Cameroon.

File: QIIME2_Workflow_Script.txt

Description: A script of a Qiime2 Workflow to process ZBJ dietary DNA. The code removes the adapters and primers, performs error correction, merging of the double stranded DNA, the creation of ASVs and taxonomic assignment.

File: Taxonomy.zip

Description: This folder contains the Qiime2 outputs from the taxonomic assignment of the 16Farms data.

File: cutadapters.txt

Description: This is the report created from using cutadapt to remove the adapters from the ZBJ dietary sequences.

File: cutZBJ.qzv

Description: This is the Qiime2 visualisation file of the dietary sequences following the removal of the ZBJ primers.

File: cutZBJ.txt

Description: This is the report created from using cutadapt to remove the primers from the ZBJ dietary sequences.

File: manifest.tsv

Description: This file contains the file paths of the dietary data required for import into Qiime2.

Variables

• sample-id:

• forward-absolute-filepath:

• reverse-absolute-filepath:

File: ZBJ_featuretable.qza

Description: The file contains information on the number of times each ASV is present in each sample.

File: ZBJ_featuretable.qzv

Description: This is a Qiime2 visualisation file of the feature table containing information on the ASV count in each sample.

File: ZBJ_rep_seqs.qza

Description: This file contains the representative unique ASV sequences present in the ZBJ dietary data.

File: ZBJ_rep_seqs.qzv

Description: This file contains a Qiime2 visualisation of the representative ASV sequences present in the ZBJ dietary data.

File: ZBJ_stats.qza

Description: This file summarises the number of reads that were retained after each DADA2 filtering step.

File: ZBJ_stats.qzv

Description: This is a Qiime2 visualisation file containing a table of the number of reads retained after each DADA2 filtering step for each sample.

File: cutZBJ.qza

Description: This is the Qiime2 file containing the ZBJ dietary sequences with the primers removed.

File: ZBJ_Sequence_Files_2.zip

Description: This file contains dietary DNA from birds and bats sampled in Cameroon.

Code/software

The software used to generate this data includes:

• High performance computing

• Miniconda3

• Qiime2-amplicon-2025.7

• R version 4.3.1

• Microsoft Excel