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.