SimPlot-CL: Automated Similarity Plotting from the Command Line

We are excited to share SimPlot-CL, a lightweight, Python-based command-line tool that enables automated similarity plot (SimPlot) analyses for multiple query sequences against a set of references.

Similarity plots are widely used in viral genomics to visualize how closely a query genome resembles a set of reference sequences along its length - for example, to explore recombination signals, genotype relationships, or mosaic genome patterns. While graphical tools such as the original SimPlot (Lole et al., 1999) and SimPlot++ (Samson et al., 2022) provide user-friendly interfaces, they can become cumbersome when running large batches of analyses.

SimPlot-CL was developed to solve exactly that problem: enabling automated similarity plotting analyses directly from the command line. The easy-to-use, open-source code behind SimPlot-CL is available on our GitHub.

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What SimPlot-CL Does

Given sets of query and reference nucleotide sequences in FASTA format, SimPlot-CL:

• Optionally aligns sequences using MAFFT
• Splits the alignment into overlapping sliding windows
• Computes pairwise similarity (1 − p-distance) between a query and reference sequences in each window
• Groups and colours sequences by genotype (if specified in metadata)
• Produces:
  • Publication-ready similarity plots (PNG, PDF, SVG, or JPG)
  • Optional CSV tables with window-by-window similarity values

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Why It’s Useful

SimPlot-CL is particularly helpful for researchers who:

• Work with large viral genome datasets
• Need to compare multiple queries against a panel of references
• Want reproducible, scriptable analyses for pipelines or HPC environments
• Prefer automation over manual GUI-based workflows

Because it runs entirely from the command line, it integrates easily into bioinformatics pipelines and allows high-throughput analyses without repetitive manual steps.

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Flexible and Customizable

The tool supports:

• Custom window and step sizes
• Different gap-handling strategies
• Separate query and reference alignments, or specifying query and reference sequences from one alignment
• Optional genotype annotations by integrating metadata
• Custom color mappings
• Optional export of similarity tables and alignments

This makes it suitable both for quick exploratory analyses and for generating figures for publications.

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Code and Documentation

The full source code, example datasets, and usage instructions are available on our GitHub.

The repository includes example commands, demo datasets, and customizable metadata/color configurations to help you get started quickly. Contributions, feedback, and feature suggestions are very welcome!

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References

Original SimPlot software: https://sray.med.som.jhmi.edu/SCRoftware/SimPlot/
Lole, Kavita S., et al. “Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination.” Journal of virology 73.1 (1999): 152-160.

SimPlot++ (modern GUI version): https://github.com/Stephane-S/Simplot_PlusPlus
Samson, Stéphane, Étienne Lord, and Vladimir Makarenkov. “SimPlot++: a Python application for representing sequence similarity and detecting recombination.” Bioinformatics 38.11 (2022): 3118-3120.

MAFFT: https://mafft.cbrc.jp/alignment/software/
Katoh, Kazutaka, and Daron M. Standley. “MAFFT multiple sequence alignment software version 7: improvements in performance and usability.” Molecular biology and evolution 30.4 (2013): 772-780.