MUSCLE(1)

💖 MUSCLE — bestie edition 💖

okay so this is the manual page for muscle, and it lives in section 1 of the manual (that's the "user commands" section, iykyk). let's get into it 🫶

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🌸 NAME 🌸

muscle — Multiple alignment program of protein sequences

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✨ DESCRIPTION ✨

So MUSCLE is a multiple alignment program for protein sequences, and yes the name is an acronym — it stands for multiple sequence comparison by log-expectation. Iconic naming behavior honestly. 💅

And like… in the authors' tests, MUSCLE achieved the highest scores of all tested programs on several alignment accuracy benchmarks, AND it's also one of the fastest programs out there. accuracy AND speed?? we love a main character who can do both. 👑

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🎀 USAGE 🎀

🦋 Align FASTA input, write aligned FASTA (AFA) output:

muscle -align input.fa -output aln.afa

🦋 Align large input using Super5 algorithm if -align is too expensive:

Use this when -align is too expensive — typically you need it once you've got more than a few hundred sequences. it's giving "big girl alignment energy" fr fr:

muscle -super5 input.fa -output aln.afa

🦋 Single replicate alignment:

muscle -align input.fa -perm PERM -perturb SEED -output aln.afa
muscle -super5 input.fa -perm PERM -perturb SEED -output aln.afa

okay so let's decode those placeholders bestie:

• PERM is the guide tree permutation, and the valid values are none, abc, acb, bca. the default is none.
• SEED is the perturbation seed, values 0, 1, 2, ... and the default is 0, which means don't perturb. no cap.

🦋 Ensemble of replicate alignments, output in Ensemble FASTA (EFA) format:

EFA has one aligned FASTA for each replicate, and the header line looks like "<PERM.SEED". yes, that's the literal shape of it. 🌈

muscle -align input.fa -stratified -output stratified_ensemble.efa
muscle -align input.fa -diversified -output diversified_ensemble.afa

• -replicates N — the number of replicates. defaults are 4, 100, 100 for stratified, diversified, and resampled respectively. and heads up: with -stratified there is one replicate per guide tree permutation, so the total is 4 x N. math is math. 🧮

🦋 Generate resampled ensemble from existing ensemble by sampling columns with replacement:

muscle -resample ensemble.efa -output resampled.efa

• -maxgapfract F — maximum fraction of gaps in a column. F goes from 0 to 1, and the default is 0.5.
• -minconf CC — minimum column confidence. CC goes from 0 to 1, and the default is 0.5.

👀 important: if the ensemble output filename has an @ in it, then one FASTA file gets generated for each replicate, where @ is replaced by perm.s. otherwise, all the replicates get written to one EFA file. choose your own adventure, bestie.

🦋 Calculate dispersion of an ensemble:

muscle -disperse ensemble.efa

🦋 Extract replicate with highest total CC:

diversified input is recommended here, just saying ✨

muscle -maxcc ensemble.efa -output maxcc.afa

🦋 Extract aligned FASTA files from EFA file:

muscle -efa_explode ensemble.efa

🦋 Convert FASTA to EFA:

the input has one filename per line, okay? one. per. line. 💖

muscle -fa2efa filenames.txt -output ensemble.efa

🦋 Update ensemble by adding two sequences of digits to each replicate:

those digits are the column confidence (CC) values — so "73" means CC=0.73, and "++" means CC=1.0. cute little shorthand, obsessed. 🍓

muscle -addconfseqs ensemble.efa -output ensemble_cc.efa

🦋 Calculate letter confidence (LC) values:

-ref specifies the alignment to compare against the ensemble (e.g. one that came from -maxcc). the output is in aligned FASTA format, except it has LC values 0, 1 ... 9 instead of letters. numbers where the letters used to be, slay. 🔥

muscle -letterconf ensemble.efa -ref aln.afa -output letterconf.afa

• -html aln.html — alignment colored by LC in HTML format. aesthetic AND informative. 🎨
• -jalview aln.features — Jalview feature file with LC values and colors. 💜

🦋 More documentation at:

https://drive5.com/muscle

go read it, bestie, it's free knowledge 🌸

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👑 AUTHOR 👑

This manpage was written by Andreas Tille for the Debian distribution, and it can be used for any other usage of the program. thank you Andreas, we appreciate you 🫶

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and that's the whole thing!! muscle 5.1, dated January 2022 — go align some proteins, you've got this 💅✨🦋