Dec 01, 2023
Nephele DADA2 Workflow
- 1North Carolina State University
Protocol Citation: Nandie Elhadidy 2023. Nephele DADA2 Workflow. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xybzlqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 01, 2023
Last Modified: December 01, 2023
Protocol Integer ID: 91672
Keywords: nephele dada2 workflow, dada2 on nephele, utilizing dada2, dada2, nephele, using amplicon sequence variant, amplicon sequence variant, pipeline, species assignment, workflow, amplified sequence, provided practice dataset
Abstract
A protocol outlining the workflow of utilizing DADA2 on Nephele with a provided practice dataset. DADA2 is a pipeline that provides species assignments using Amplicon Sequence Variants or ASVs which compare variants to a predetermined sequence through assumption that the most amplified sequence is the correct one.
Downloading the Dataset
To download the practice dataset provided by Nephele, visit: https://nephele.niaid.nih.gov/user_guide_data/
Download the first set of sequences listed as well as the mapping file.
The reference provided for this set of sequences is listed as: Experimental Microbial Dysbiosis Does Not Promote Disease Progression in SIV-Infected Macaques. NCBI BioProject: PRJNA417022
Once downloaded, unzip the downloaded file and ensure all .fastq.gz files are present.
Running DADA2
Click the Home tab.
Choose the Analyze option.
Under Amplicon Metagenomics, select "Start job" option under DADA2.
Select Paired End FASTQ, as this is a paired-end dataset.
Click "Upload from my computer" and add all .fastq.gz files from the downloaded dataset. Start upload
Select "Next" and upload the corresponding Mapping File.
After reaching the Pipeline Selection page, click Select and enter a description of the job.
Keep Filter and Trim, Merge Pairs, and Analysis settings defaulted. Do not check Ion Torrent Data. Click Validate and Submit *Note: This is for following the exact tutorial instructions. If you would like to make changes to these pre-sets, proceed with these changes.
Nephele will send an email upon job completion.
Analysis of the Output
From the My Workspace tab, hover to the view results icon under Actions for the completed DADA2 job.
Under Reported Statistics, Reported Files, Reported Images, Reported Tables, and Reported Charts and explore outputs.
Reported files of note include: Rooted tree, Rarefaction curve, Alpha diversity, PCoA binomial, BCoA Bray-Curtis, and ASV heatmap.
Reported images of note include: Quality profiles of forward and reverse reads
Reported tables of note include: Read counts by sample and Top species table
Reported charts of note include: Reads in vs. reads out and Track reads
Protocol references