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>: 03/08/2025<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 Cameroon.Qiime2 bioinformatic output files including the final 16-farms-ASV table.An 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

如何从动物粪便样本开展饮食DNA宏条形码（DNA Metabarcoding）研究：数据与文件结构说明

**项目：** 如何从动物粪便样本开展饮食DNA宏条形码研究
**日期：** 2025年8月3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：** 本数据集可用于运行示例生物信息学工作流，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个农场的鸟类和蝙蝠样本中采集的277份样本的饮食测序数据。
2. Qiime2生物信息学输出文件：包括最终的16-farms-ASV表。
3. NCBI 细胞色素c氧化酶亚基I（Cytochrome c Oxidase Subunit I, COI）ZBJ数据库与分类器（classifier）。
4. 统计分析相关文件：包含鸟类和蝙蝠的元数据（metadata）、用于清理和整理饮食数据的R脚本、用于构建基础广义线性模型（Generalized Linear Model, GLM）、多变量分析以及网络分析的代码，同时包含脚本生成的图表与结果。

## 文件与变量
### 文件：Statistical_Analysis_Files.zip
**描述：** 该文件夹包含鸟类和蝙蝠饮食扩增子序列变异体（Amplicon Sequence Variant, ASV）表"16Farms_ASV_Table.csv"，以及元数据文件"bird_and_bat_capturedata.csv"和"faeces_sample_database.xlsx"。文件夹中还包含用于统计分析的R脚本："1.organize&clean_metabarcoding.R"（用于过滤并整理用于统计分析的ASV表）、"2.glm_examples"（包含广义线性模型与多变量分析的代码）、"3.building_analysing_networks"（包含饮食信息的网络分析代码）。缺失值以"NA"表示。

### 文件：NCBI_Database.zip
**描述：** 该文件夹包含通过RESCRIPt从NCBI下载的COI参考序列与分类学信息，以及本教程（https://forum.qiime2.org/t/using-rescripts-extract-seq-segments-to-extract-reference-sequences-without-pcr-primer-pairs/23618）生成的输出文件（Robeson et al., 2021; Rognes et al., 2016）。同时包含由Qiime2基于ZBJ参考序列与分类学信息构建的分类器"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：** 用于处理ZBJ饮食DNA的Qiime2工作流脚本，代码可移除接头与引物、完成错误校正、双链DNA合并、扩增子序列变异体（ASV）构建以及分类学注释。

### 文件：Taxonomy.zip
**描述：** 该文件夹包含对16Farms数据进行分类学注释后的Qiime2输出结果。

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

### 文件：cutZBJ.qzv
**描述：** 移除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移除ZBJ饮食序列引物后生成的报告。

### 文件：manifest.tsv
**描述：** 包含导入Qiime2所需的饮食数据文件路径的文件。
#### 变量
sample-id:forward-absolute-filepath:reverse-absolute-filepath:

### 文件：ZBJ_featuretable.qza
**描述：** 该文件包含每份样本中各ASV的出现次数信息。

### 文件：ZBJ_featuretable.qzv
**描述：** 包含每份样本中ASV计数信息的特征表的Qiime2可视化文件。

### 文件：ZBJ_rep_seqs.qza
**描述：** 该文件包含ZBJ饮食数据中存在的代表性独特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：** 该文件汇总了每一轮DADA2过滤步骤后保留的读数数量。

### 文件：ZBJ_stats.qzv
**描述：** 包含每份样本在每一轮DADA2过滤步骤后保留的读数数量表格的Qiime2可视化文件。

### 文件：cutZBJ.qza
**描述：** 该Qiime2文件包含已移除引物的ZBJ饮食序列。

### 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
本数据集生成所使用的软件包括：
高性能计算集群、Miniconda3、Qiime2-amplicon-2025.7、R 4.3.1版本、Microsoft Excel