First download and install Muscle v5.
To make an alignment, use the align command, like this:
muscle -align sequences.fasta -output alignment.fasta
If you just want one, high-quality MSA, and that worked for you, that's it, you're done!
If your sequence set was so big that the align command took too much time or memory, try the super5 command instead.
How to check if your alignment has errors
Make a stratified ensemble by using the -stratified option of align, like this:
muscle -align sequences.fasta -stratified -output ensemble.efa
This will create an ensemble FASTA (EFA) file containing 16 alignments. Measure the dispersion of the ensemble like this:muscle -disperse ensemble.efa
If the dispersion is zero, then all the MSAs in the ensemble are the same and your alignment is robust. Quite likely, it has no errors. If you see a large dispersion, say bigger than 0.05, then there is significant variation between the alignments, and this is necessarily explained by alignment errors. You should then start to consider whether these differences affect your downstream analysis, e.g. making trees. You can check this by doing the analysis separately for each replicate.
