Alignment and phylogenetics · Software
MUSCLE
Generates multiple sequence alignments for proteins and nucleotides, alongside protein structure alignments.
What MUSCLE is
MUSCLE is software developed by Robert C. Edgar for constructing multiple sequence alignments of biological macromolecules, including proteins and nucleotides. It also supports multiple protein structure alignments via MUSCLE-3D.
A central problem in sequence analysis is the downstream sensitivity of biological conclusions to alignment ambiguities and errors. MUSCLE addresses this by generating ensembles of alternative alignments that retain high accuracy, allowing researchers to measure how robust phylogenetic trees or homology predictions are to alignment variability.
The software is designed to scale to thousands of sequences or structures on standard desktop computers, achieving top benchmark scores on Balibase, Bralibase, and Balifam test suites.
How it works
MUSCLE constructs multiple alignments using algorithms such as the default align method or the Super7 method for larger datasets. To support structure alignment (MUSCLE-3D), it processes intermediate 'mega' files converted from structural data alongside distance matrices created by the companion tool reseek.
Key facts
- Current version
- 5.3
- Supported inputs
- Protein sequences, nucleotide sequences, and mega structure files generated by reseek
- Supported outputs
- Aligned fasta (.afa) files and alignment ensembles
- Benchmark validation
- Balibase, Bralibase, and Balifam
What you can use it for
- Protein and nucleotide sequence alignment
- Aligning biological sequences across large sets of proteins or nucleotides on standard computing hardware.
- Evaluating phylogenetic robustness
- Generating alignment ensembles with equivalent accuracy scores to evaluate whether downstream phylogenetic trees are resilient to alignment variation.
- Scalable multiple structure alignment
- Aligning up to 10,000 protein structures in combination with distance matrices and mega files generated by reseek.
Run it here
MUSCLE is preinstalled on the machine behind every research conversation. Ask the agent in plain language, or give it the exact command — it runs it, reads the output and explains it.
Try asking
“Make a multiple sequence alignment of these sequences with MUSCLE.”
Strengths and limits
Good at
- Achieves top scores on Balibase, Bralibase, and Balifam benchmark datasets.
- Generates ensembles of alternative alignments with accuracy matching default parameters to assess alignment uncertainty.
- Scales to thousands of sequences or protein structures on a standard desktop computer.
- Runs as a self-contained binary with no external software dependencies.
Watch out for
- Performing protein structure alignments requires preprocessing structures into intermediate formats using the external reseek software.
- Scaling structure alignments up to 10,000 structures requires generating an explicit distance matrix.
Questions
Can I run MUSCLE online without installing it?
MUSCLE is preinstalled on the machine behind every research conversation. Ask the agent in plain language, or give it the exact command — it runs it, reads the output and explains it.
What licence is MUSCLE under?
MUSCLE is published under its authors' own licence; the project page at https://drive5.com/muscle/ states it.
What types of molecules can MUSCLE align?
MUSCLE aligns protein and nucleotide sequences, as well as multiple protein structures using MUSCLE-3D.
What is an alignment ensemble in MUSCLE?
An ensemble is a collection of alternative alignments generated with comparable accuracy to evaluate how much alignment variation impacts downstream analysis such as phylogeny.
How are protein structures prepared for MUSCLE-3D?
Structures are preprocessed using the pdb2mega or convert commands in the reseek tool to create .mega files and distance matrices that MUSCLE accepts as input.
How to cite it
Edgar RC., Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nature Communications 13.1 (2022): 6968. https://www.nature.com/articles/s41467-022-34630-w.pdf
Written from MUSCLE's own README and project page. Statements citing a figure that does not appear there are removed before publication. Licence and running details come from our registry.