How do matrix weights for amino acid 'X' affect protein alignments in MUSCLE?

[hepcat72]

Sorry, this is a cross-post from biostars, but I haven't had any luck there in figuring this out...

I'm developing a protein weight matrix from scratch to use with MUSCLE. I started with the 20 amino acids and the stop character, but using just those, I get a warning from MUSCLE that makes no sense^. But if I add a row & column for the amino acid code 'X' (with all 0s - everything else ranges from 1-25), the warning goes away. However, even though the sequences I'm aligning have no occurrences of 'X', the alignments produced using the matrix without 'X' and the matrix with 'X' are different. I don't understand why that is.

How do alignment algorithms treat 'X' in the weight matrix when there are no X's in the sequences being aligned? Why would an alignment produced using a matrix with 'X' versus an alifgnment produced using a matrix without 'X' be different? Are there weights I can insert for 'X' in the matrix that would not affect the alignment?

Side question - I don't have 'B' or 'Z' in my matrix either (nor do they occur in my sequences). Do they need to be in the matrix for the alignment software to yield good results?

^ [I]The warning from muscle is "*** WARNING *** Matrix is not symmetrical, ?->?=5, ?->?=0". This doesn't make sense for 3 reasons: 1. the matrix is symmetrical. 2. There were no 0s in my matrix when the warning occurred. 3. Simply adding a row/column for 'X' silenced the warning. I looked briefly at the code in muscle where the warning is generated and it's simply checking that

matrix[i][j] == matrix[j][i][/i]

. The fact that there's an item that doesn't match is unsettling. Perhaps this is a hidden requirement that 'X' must be present in the matrix - but still, why would the alignment change with/without it?

[hepcat72]

The answer is, the presence or absence of 'X' in a protein weight matrix should not affect the alignment of sequences not containing 'X', however there is a bug in muscle that is fixed by the following patch. Supplying muscle a custom weight matrix without any of the ambiguous amino acids causes the alignment to differ because muscle was not setting the weights in the last column and that was causing the warnings.

https://github.com/Bioconductor-mirror/muscle/compare/master...hepcat72atch-2

Sorry, this is a cross-post from biostars, but I haven't had any luck there in figuring this out...

I'm developing a protein weight matrix from scratch to use with MUSCLE. I started with the 20 amino acids and the stop character, but using just those, I get a warning from MUSCLE that makes no sense^. But if I add a row & column for the amino acid code 'X' (with all 0s - everything else ranges from 1-25), the warning goes away. However, even though the sequences I'm aligning have no occurrences of 'X', the alignments produced using the matrix without 'X' and the matrix with 'X' are different. I don't understand why that is.

How do alignment algorithms treat 'X' in the weight matrix when there are no X's in the sequences being aligned? Why would an alignment produced using a matrix with 'X' versus an alifgnment produced using a matrix without 'X' be different? Are there weights I can insert for 'X' in the matrix that would not affect the alignment?

Side question - I don't have 'B' or 'Z' in my matrix either (nor do they occur in my sequences). Do they need to be in the matrix for the alignment software to yield good results?

^ [I]The warning from muscle is "*** WARNING *** Matrix is not symmetrical, ?->?=5, ?->?=0". This doesn't make sense for 3 reasons: 1. the matrix is symmetrical. 2. There were no 0s in my matrix when the warning occurred. 3. Simply adding a row/column for 'X' silenced the warning. I looked briefly at the code in muscle where the warning is generated and it's simply checking that

matrix[i][j] == matrix[j][i][/i]

. The fact that there's an item that doesn't match is unsettling. Perhaps this is a hidden requirement that 'X' must be present in the matrix - but still, why would the alignment change with/without it?