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I applied three multiple sequence alignment methods of muscle, mafft and clistal omega on 30 sequences and 400 sequences . In the case of 30 seq, the output of alignment from the 3 methods was in the same length but in the case of larger data with different length of sequences (input data) the output from alignment methods was different length clustal length is different than the two others. My question is how can i assure that the length from MSA should be equal or different?

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The length of the alignment will depend on the model. For example, high gap costs will typically result in shorter alignments, whereas low gap-costs will do the opposite.

Consider the sequences below:

AAAAA
TTTTT
GGGGG

with a very gap cost, these sequences will result in the following MSA

AAAAA
|||||
TTTTT
|||||
GGGGG

but of course with lower gap costs, we'd observe

AAAAA----------
-----TTTTT-----
----------GGGGG

Of course, this is an extreme case, but it goes to show that the alignment model will vary how long the resulting alignment is.

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    $\begingroup$ I noticed also one thing when the input (length of sequences) before the MSA method are on the same length, the output after alignment methods are all on equal length, however, when the input differs, the output differs as well. Is it right? $\endgroup$ Oct 8 at 22:41
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    $\begingroup$ For a single run of MSA on a set of sequences, the output lengths should all be the same, regardless of input length. $\endgroup$ Oct 9 at 1:27

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