2 Mothur Microbial Profiling

2.1 Tutorial References

Before diving into the Mothur bioinformatics pipeline, make sure to familiarize yourself with the following essential references:

1. Snakemake Workflow: Explore the comprehensive Snakemake workflow designed to run Mothur within a conda environment. So far, we have found this repository to be a great demo for maintaining reproducibility using Snakemake workflow, making it a good starting point. However, users are encouraged to explore more elsewhere to see if there is a better alternative.

2. Full Mothur Tutorial: Refer to the official Mothur tutorial for a detailed guide on utilizing Mothur in bioinformatics analysis.

2.2 Setting Up Mothur in a Conda Environment

2.2.1 Clone the Snakemake Workflow

I strongly suggest using a repository that offers a comprehensive guide on how to integrate Mothur workflows using Snakemake. To access this incredibly helpful resource, I recommend cloning the Snakemake workflow repository onto your computer. The repository presents a detailed explanation of each step involved in the process, ensuring a smooth implementation of Mothur within the Snakemake framework.

git clone https://github.com/wclose/mothurPipeline.git

2.3 Navigate to the Workflow Directory:**

• Change into the cloned directory: bash cd mothurPipeline

3. Set Up Conda Environment:

   • Create a conda environment for Mothur:

     conda env create -f environment.yaml

4. Activate Conda Environment:

   • Activate the conda environment:

     conda activate mothur

5. Run the Snakemake Workflow:

   • Execute the Snakemake workflow to start the Mothur analysis:

     snakemake --use-conda

These steps provide a simplified entry point to using Mothur within a conda environment. For detailed explanations and additional features, refer to the full Snakemake workflow repository.

2.4 Create a mothur YAML file

name: mothur48
channels:
    - conda-forge
    - bioconda
    - defaults
dependencies:
    - mothur =1.48.0
    - vsearch =2.22.1

2.5 Create mothur environment using YAML File

conda activate base
conda env create -n mothur48 --file mothur48.yml
conda activate mothur48 

2.6 Download References Databases

• Download Silva alignment reference database.
• Optionally create Silva classifier from Silva alignments.
• Download RDP classifier.
• Get any other suitable classifier compatible with Mothur.

The download script is in the repo, saved in the workflow/scripts directory. Run the following on command line or use the snakemake rules.

bash workflow/scripts/mothurReferences.sh