Non-Reversibility of the Evolutionary Process (NRM)

NRM (Non-Reversibility of the Evolutionary Process) is a statistical method used to test the non-reversibility of nucleotide substitutions in evolutionary models. This approach helps researchers to identify whether the evolutionary process at certain sites in a DNA sequence is reversible or not, providing vital insights into molecular evolution and the underlying selection pressures that shape genetic diversity.

Citation

If you use NRM in your analysis, please cite:

Kosakovsky Pond, S.L., & Frost, S.D.W. (2005). "Not So Different After All: A Comparison of Methods for Detecting Amino Acid Sites Under Selection." Molecular Biology and Evolution.

Input Parameters

Required Inputs

• Alignment File: A fasta or other supported format file containing the sequence alignments for the analysis.
• Phylogenetic Tree: A file representing the phylogenetic relationships (newick format preferred).
• Genetic Code: The genetic code to apply (default is "Universal").

Optional Inputs

• Rate Classes: The number of classes for rates (default: 1).
• Triple Islands: Specify whether to use triple islands (default: "No").
• Output File: An option to specify a filename for the output results.

Outputs

Summary

NRM produces a JSON file containing comprehensive analysis details, including but not limited to:

• Metadata about the analysis and its parameters.
• Site-specific results detailing non-reversible substitution rates.
• A descriptive table summarizing key statistics of the evolutionary process across sequences.

Site-Level Output Details

The site-level output includes the following:

• Transition Rates: Estimates of transition probabilities between nucleotides.
• Statistical Significance: P-values assessing the likelihood of reversibility.
• Selection Pressure Analysis: Insight into selective forces acting upon the sequences tested.

Visualization

An interactive visualization tool is available to help users explore the results effectively. This tool provides:

• Summary Statistics: Visualization of the number of sequences and sites analyzed.
• Matrix Displays: Graphical representations of transition rates for quick insight into the evolutionary patterns.
• Interactive Charts: Plots for detailed examination of substitution dynamics across various sites.

Example Workflow

Follow these steps to effectively use the NRM tool through the website:

1. Upload Data:

   • Navigate to the NRM page.
   • Upload the alignment file and phylogenetic tree file.
   • Specify the genetic code and any optional parameters.

2. Run Analysis:

   • Click the "Run Analysis" button to start the NRM analysis.

3. Review Results:

   • Once the analysis completes, navigate to the results page via the provided link.
   • Explore summary statistics, visualizations, and detailed results tables.

4. Export Results:

   • Download the JSON results file for external analysis or archiving as needed.

Example Usage

To run the NRM analysis using HyPhy, you can use the following commands.

Full Example Command

NRM.bf is located at https://github.com/veg/hyphy-analyses/tree/master/NucleotideNonREV

/path/to/hyphy/hyphy NRM.bf \
  --alignment /path/to/alignment_file.fasta \
  --tree /path/to/tree_file.newick \
  --genetic_code Universal \
  --rate_classes 2 \
  --output /path/to/output.json

Parameters

• --alignment: Sequence alignment to fit non-reversible models to (required).
• --tree: A phylogenetic tree (optionally annotated with {}) (required).
• --genetic_code: The genetic code to apply (default is "Universal").
• --rate_classes: The number of classes for rates (default: 1).
• --output: Write the resulting JSON to this file (default is to save to the same path as the alignment file + 'NRM.json').

Minimal Example Command

/path/to/hyphy/hyphy NRM.bf \
  --alignment /path/to/alignment_file.fasta \
  --tree /path/to/tree_file.newick