How to do Diet DNA Metabarcoding from Animal Faecal Samples

How to do Diet DNA Metabarcoding from Animal Faecal SamplesDescription of the data and file structure<b>Project:</b> How to do Diet DNA Metabarcoding from Animal Faecal Samples<b>Date</b>: 30/08/2025<b>Authors: Rachel D. McConnell and Crinan Jarrett, Diogo F. Ferreira, Luke L. Powell, Alma L.S. Quiñones, Davide M. Dominoni, Andreanna J. Welch</b><b>Purpose:</b> 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:ZBJ_Sequence_files containing dietary sequencing data from 277 samples collected from birds and bats across 16 farms in CameroonQiime2 bioinformatic output files including the final 16-farms-ASV tableAn NCBI COI ZBJ database and classifierStatistical 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.<b>Files and variables</b><b>File: Statistical_Analysis_Files.zip</b><b>Description:</b> This folder contains the bird and bat dietary ASV Table "16Farms_ASV_Table.csv" and the<br>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&amp;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".<br><b>File: NCBI_Database.zip</b><b>Description:</b> 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/using-rescripts-extract-seq-segments-to-extract-reference-sequences-without-pcr-primer-pairs/23618 (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".<b>File: ZBJ_Sequence_Files_1.zip</b><b>Description:</b> This file contains dietary DNA from birds and bats sampled in Cameroon.<b>File: QIIME2_Workflow_Script.txt</b><b>Description:</b> 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.<b>File: Taxonomy.zip</b><b>Description:</b> This folder contains the Qiime2 outputs from the taxonomic assignment of the 16Farms data.<b>File: cutadapters.txt</b><b>Description:</b> This is the report created from using cutadapt to remove the adapters from the ZBJ dietary sequences.<b>File: cutZBJ.qzv</b><b>Description:</b> This is the Qiime2 visualisation file of the dietary sequences following the removal of the ZBJ primers.<b>File: cutZBJ.txt</b><b>Description:</b> This is the report created from using cutadapt to remove the primers from the ZBJ dietary sequences.<b>File: manifest.tsv</b><b>Description:</b> This file contains the file paths of the dietary data required for import into Qiime2.<b>Variables</b>sample-id:forward-absolute-filepath:reverse-absolute-filepath:<b>File: ZBJ_featuretable.qza</b><b>Description:</b> The file contains information on the number of times each ASV is present in each sample.<b>File: ZBJ_featuretable.qzv</b><b>Description:</b> This is a Qiime2 visualisation file of the feature table containing information on the ASV count in each sample.<b>File: ZBJ_rep_seqs.qza</b><b>Description:</b> This file contains the representative unique ASV sequences present in the ZBJ dietary data.<b>File: ZBJ_rep_seqs.qzv</b><b>Description:</b> This file contains a Qiime2 visualisation of the representative ASV sequences present in the ZBJ dietary data.<b>File: ZBJ_stats.qza</b><b>Description:</b> This file summarises the number of reads that were retained after each DADA2 filtering step.<b>File: ZBJ_stats.qzv</b><b>Description:</b> This is a Qiime2 visualisation file containing a table of the number of reads retained after each DADA2 filtering step for each sample.<b>File: cutZBJ.qza</b><b>Description:</b> This is the Qiime2 file containing the ZBJ dietary sequences with the primers removed.<b>File: ZBJ_Sequence_Files_2.zip</b><b>Description:</b> This file contains dietary DNA from birds and bats sampled in Cameroon.<b>Code/software</b>The software used to generate this data includes:High performance computingMiniconda3Qiime2-amplicon-2025.7R version 4.3.1Microsoft Excel<br>

# 动物粪便样本饮食DNA宏条形码分析实操指南
## 数据集与文件结构说明
**项目：** 动物粪便样本饮食DNA宏条形码分析实操指南
**日期：** 2025年8月30日
**作者：** Rachel D. McConnell、Crinan Jarrett、Diogo F. Ferreira、Luke L. Powell、Alma L.S. Quiñones、Davide M. Dominoni、Andreanna J. Welch
**用途：** 本数据集文件可用于运行示例生物信息学分析流程，并对鸟类和蝙蝠的宏条形码饮食DNA开展统计分析。数据集包含以下内容：
1. ZBJ_Sequence_files：包含从喀麦隆16个养殖场采集的277份鸟类和蝙蝠样本的饮食测序数据
2. Qiime2生物信息学输出文件：包含最终的16个养殖场扩增子序列变体（ASV）表
3. NCBI COI ZBJ数据库与分类器
4. 统计分析相关文件：包含鸟类和蝙蝠的元数据、用于清理和整理饮食数据的R脚本、可执行基础广义线性模型、多变量分析及网络分析的代码，同时包含脚本生成的图表与结果。

**文件与变量说明**
**文件：Statistical_Analysis_Files.zip**
**描述：** 该压缩包包含鸟类和蝙蝠饮食ASV表"16Farms_ASV_Table.csv"、元数据文件"bird_and_bat_capturedata.csv"与"faeces_sample_database.xlsx"，同时包含用于统计分析的R脚本：包括用于过滤并整理ASV表以开展统计分析的"1.organize&clean_metabarcoding.R"、用于执行广义线性模型与多变量分析的"2.glm_examples"，以及用于开展饮食信息网络分析的"3.building_analysing_networks"。缺失值以"NA"标注。

**文件：NCBI_Database.zip**
**描述：** 该压缩包包含通过RESCRIPt从NCBI下载的COI参考序列与分类学信息，以及基于以下教程生成的输出文件：https://forum.qiime2.org/t/using-rescripts-extract-seq-segments-to-extract-reference-sequences-without-pcr-primer-pairs/23618（Robeson et al., 2021; Rognes et al., 2016）。同时包含通过Qiime2基于ZBJ参考序列与分类学信息构建的COI分类器"zbj-ncbi-classifier.qza"。

**文件：ZBJ_Sequence_Files_1.zip**
**描述：** 该文件包含从喀麦隆采样的鸟类和蝙蝠的饮食DNA。

**文件：QIIME2_Workflow_Script.txt**
**描述：** Qiime2分析流程脚本，用于处理ZBJ饮食DNA序列，代码可完成接头与引物切除、错误校正、双链DNA合并、ASV生成及分类学注释。

**文件：Taxonomy.zip**
**描述：** 该压缩包包含对16Farms数据进行分类学注释后生成的Qiime2输出文件。

**文件：cutadapters.txt**
**描述：** 使用cutadapt切除ZBJ饮食序列接头后生成的报告文件。

**文件：cutZBJ.qzv**
**描述：** 切除ZBJ引物后的饮食序列的Qiime2可视化文件。

**文件：cutZBJ.txt**
**描述：** 使用cutadapt切除ZBJ饮食序列引物后生成的报告文件。

**文件：manifest.tsv**
**描述：** 包含导入Qiime2所需的饮食数据文件路径信息，变量包括：sample-id、forward-absolute-filepath、reverse-absolute-filepath。

**文件：ZBJ_featuretable.qza**
**描述：** 包含每个样本中各ASV的出现次数信息。

**文件：ZBJ_featuretable.qzv**
**描述：** 包含各样本ASV计数信息的特征表的Qiime2可视化文件。

**文件：ZBJ_rep_seqs.qza**
**描述：** 包含ZBJ饮食数据中代表性的唯一ASV序列。

**文件：ZBJ_rep_seqs.qzv**
**描述：** 包含ZBJ饮食数据中代表性ASV序列的Qiime2可视化文件。

**文件：ZBJ_stats.qza**
**描述：** 汇总了每个DADA2过滤步骤后保留的测序读段数量。

**文件：ZBJ_stats.qzv**
**描述：** 包含每个样本在各DADA2过滤步骤后保留的测序读段数量表格的Qiime2可视化文件。

**文件：cutZBJ.qza**
**描述：** 包含已切除引物的ZBJ饮食序列的Qiime2文件。

**文件：ZBJ_Sequence_Files_2.zip**
**描述：** 该文件包含从喀麦隆采样的鸟类和蝙蝠的饮食DNA。

**代码与软件**
生成本数据集所用的软件包括：高性能计算集群、Miniconda3、Qiime2-amplicon-2025.7、R版本4.3.1、Microsoft Excel。