QIIME2 pipeline for ITS2 based "nemabiome" sequencing in veterinary species

This repository contains resources and example scripts for running a QIIME 2 workflow for nemabiome ITS2 amplicon analysis described in this article: Jesudoss Chelladurai, J., Abraham, A., Quintana, T. QIIME2 pipeline for ITS2-based nemabiome sequencing in veterinary species and the importance of analysis parameters. Parasites and Vectors. 2025. DOI: 10.1186/s13071-025-07184-1. Repository contents Six downloadable zip files are provided. 1. QIIME2_scripts.zip includes the following components: nemabiome_new_classifier.qzaA QIIME 2–formatted classifier for nemabiome ITS2 data. This classifier is the QIIME-compatible version of Nematode_ITS2_1.0.0_idtaxa.tax and is intended for use with the taxonomic assignment steps in this workflow. pe-33-manifest_exampleAn example paired-end manifest file that researchers can use as a template for preparing their own sequencing run metadata. This is a tab limited text file with the headers: "sample-id", "forward-absolute-filepath" and "reverse-absolute-filepath" separated by tabs. QIIME2_nemabiome_Part1.sh and QIIME2_nemabiome_Part2.shTwo shell scripts containing the primary QIIME 2 workflow steps. These scripts were written for QIIME2/2024.10.If you are using another QIIME 2 version, you must edit the module load command within the .sh files to match the version of QIIME2 that you have installed. sample-metadata.tsvAn example metadata file that must be customized for each study. 2. Rdada2_pipeline_nemabiome.Rmd.zip includes the following component: Rdada2_pipeline_nemabiome.RmdThis Rmd provides an R Markdown workflow for processing nemabiome ITS2 sequencing data using DADA2 in R and implements the default parameters and settings described on the nemabiome.ca website. 3. Three simulated nemabiome ITS2 sequencing datasets: Simulated_ruminant_FASTQs.zip Simulated_canine_FASTQs.zip Simulated_equine_FASTQs.zip Each .zip dataset contains 9 simulated samples, with forward and reverse reads for every sample, for a total of 18 FASTQ files per dataset. The exact compositional breakdown for each sample is provided in the accompanying publication. Please refer to the article for detailed proportions and simulation design. 4. Multifasta_for_ART_Illumina.zip includes the following components: Three FASTA files comprising ITS2 sequences obtained from the Nematode_ITS2_1.0.0_idtaxa reference set. These files were used as input for ART Illumina to generate the simulated nemabiome datasets (ruminant, canine, and equine) provided in the .zip files above. Instructions for using QIIME2 for nemabiome 1. Install QIIME2: Install the latest version of QIIME2 on your computer. If using a high-performance computing cluster, contact the HPC administrators. Make a note of the QIIME2 version that was installed to ensure the scripts below run correctly. You may need to modify a few lines of code in the provided scripts to match the version of QIIME2 that you have installed. 2. Prepare your "manifest file" and metadata: Before running any scripts: Edit the paired-end manifest file (pe-33-manifest_example) to reflect the absolute paths to your raw FASTQ files. Edit the sample-metadata.tsv file to match your samples. The sample-id values in the manifest and metadata MUST match exactly. Failure to do so will cause errors during the import and downstream steps. 3. Move the classifier nemabiome_new_classifier.qza to the same folder as the two script files. This file is required for taxonomic assignment within the workflow and corresponds to the Nematode_ITS2_1.0.0_idtaxa reference set curated for nemabiome studies. Without the classifier, the output will be incomplete. 4. Run the scripts in order Enter a command line terminal and navigate to the location of the files and downloaded scripts. Ensure that the scripts ending in ".sh" are executable by using chmod +x QIIME2_nemabiome_Part1.sh and chmod +x QIIME2_nemabiome_Part2.sh in a linux terminal. Run the scripts using ./your_script_name.sh or sh your_script_name.sh or bash your_script_name.sh depending on your command line terminal. If you are running these scripts on a high-performance computing cluster, commands to run scripts in a queue may be pre-programmed. Consult the HPC documentation or contact the HPC administrators. Once QIIME2_nemabiome_Part1.sh is successfully completed, the script will generate the file demux-trimmed.qzv in the same folder. You must open this file (demux-trimmed.qzv) in QIIME2View (https://view.qiime2.org) to examine the quality plots and to determine appropriate DADA2 parameters for trimming and truncation. Use the plots in QIIME2View to determine the 3' and 5' positions at which quality scores are too low. Do not proceed to Part 2 without completing this inspection step. Using the parameters in the previous step, edit the following lines in QIIME2_nemabiome_Part2.sh: --p-trim-left-f --p-trim-left-r --p-trunc-len-f --p-trunc-len-r Run the second script (QIIME2_nemabiome_Part2.sh) and examine the outputs. If you use these scripts or datasets, please cite our paper.本仓库包含用于开展线虫组(nemabiome)ITS2扩增子分析的QIIME 2工作流所需的资源与示例脚本，相关工作流已在以下论文中描述： Jesudoss Chelladurai, J., Abraham, A., Quintana, T. 适用于兽医物种的基于ITS2的线虫组测序QIIME2分析流程及分析参数的重要性. 《寄生虫与媒介》. 2025. DOI: 10.1186/s13071-025-07184-1. ## 仓库内容 本仓库提供六个可下载的压缩包文件。 1. QIIME2_scripts.zip 包含以下组件： - nemabiome_new_classifier.qza：一款适配QIIME 2格式的线虫组ITS2数据分类器，该分类器是Nematode_ITS2_1.0.0_idtaxa.tax的QIIME兼容版本，用于本工作流的分类学注释步骤。 - pe-33-manifest_example：一款双端样本清单(manifest)文件示例，研究人员可将其作为模板制备自己的测序运行元数据。该文件为制表符分隔的文本文件，表头为"sample-id"、"forward-absolute-filepath"与"reverse-absolute-filepath"，各字段以制表符分隔。 - QIIME2_nemabiome_Part1.sh与QIIME2_nemabiome_Part2.sh：两个Shell脚本，包含QIIME 2工作流的主要分析步骤。这些脚本针对QIIME2/2024.10版本编写。若使用其他QIIME 2版本，需修改.sh文件中的module load命令，以匹配您安装的QIIME 2版本。 - sample-metadata.tsv：一款适用于每项研究的自定义元数据示例文件。 2. Rdada2_pipeline_nemabiome.Rmd.zip 包含以下组件： - Rdada2_pipeline_nemabiome.Rmd：该R Markdown文档提供了一套使用R语言中DADA2(DADA2)算法处理线虫组ITS2测序数据的工作流，并实现了nemabiome.ca网站上描述的默认参数与设置。 3. 三套模拟线虫组ITS2测序数据集： - Simulated_ruminant_FASTQs.zip - Simulated_canine_FASTQs.zip - Simulated_equine_FASTQs.zip 每个压缩包数据集包含9个模拟样本，每个样本均配有正向与反向测序读段，单套数据集总计18个FASTQ文件。每个样本的精确组成比例已在配套论文中给出，请参阅原文获取详细的组分占比与模拟实验设计方案。 4. Multifasta_for_ART_Illumina.zip 包含以下组件： - 三套FASTA文件，包含取自Nematode_ITS2_1.0.0_idtaxa参考集的ITS2序列。这些文件被用作ART Illumina的输入文件，用于生成上述压缩包中提供的模拟线虫数据集（反刍动物、犬与马来源）。 ## 线虫组分析QIIME 2使用指南 1. 安装QIIME 2： - 在您的计算机上安装最新版本的QIIME 2。 - 若使用高性能计算(HPC)集群，请联系集群管理员。 - 记录您安装的QIIME 2版本，以确保后续脚本可正常运行。您可能需要修改提供的脚本中的少量代码行，以匹配您所安装的QIIME 2版本。 2. 准备样本清单文件与元数据： 在运行任何脚本之前： - 编辑双端样本清单文件（pe-33-manifest_example），将其修改为您的原始FASTQ文件的绝对路径。 - 编辑sample-metadata.tsv文件，使其与您的实验样本匹配。 - 样本清单与元数据中的sample-id字段值必须完全一致，否则将在导入与后续分析步骤中引发错误。 3. 将分类器文件nemabiome_new_classifier.qza移动至与两个脚本文件相同的目录下。 该文件为本工作流中分类学注释步骤的必需文件，对应为线虫研究定制的Nematode_ITS2_1.0.0_idtaxa参考集。若缺少该分类器，分析输出将不完整。 4. 按顺序运行脚本： - 打开命令行终端，切换至存放文件与下载脚本的目录。 - 在Linux终端中执行`chmod +x QIIME2_nemabiome_Part1.sh`与`chmod +x QIIME2_nemabiome_Part2.sh`命令，确保.sh后缀的脚本具备可执行权限。 - 根据您使用的命令行终端，通过`./your_script_name.sh`、`sh your_script_name.sh`或`bash your_script_name.sh`运行脚本。若在高性能计算集群上运行这些脚本，队列运行脚本的命令可能已预配置，请参阅集群文档或联系集群管理员。 - 当QIIME2_nemabiome_Part1.sh成功运行完成后，该脚本将在当前目录下生成demux-trimmed.qzv文件。 - 您必须在QIIME2View（https://view.qiime2.org）中打开该demux-trimmed.qzv文件，查看质量分布图，以确定适用于DADA2的修剪与截短参数。通过QIIME2View中的图表确定质量分数过低的3'端与5'端位置。未完成该检查步骤前，请勿运行Part 2脚本。 - 基于上一步确定的参数，编辑QIIME2_nemabiome_Part2.sh中的以下行： --p-trim-left-f --p-trim-left-r --p-trunc-len-f --p-trunc-len-r - 运行第二个脚本（QIIME2_nemabiome_Part2.sh）并查看分析输出结果。 若您使用本仓库的脚本或数据集，请引用我们的论文。