I am working with multiple alignment in ClustalX and I am assessing the first round of the alignment. I have noticed that there are few sequences where the length of the gap that were extended were quite long between the residues. Therefore my question is - how many gaps or how long should the extension be between the residues? I am certain that I have to delete the sequences with very long gap extensions and then realign but I cannot recall the gap length that is allowed.
You can influence this by optimising the Gap Opening and Gap Extension Penalties
"The GAP OPENING and EXTENSION PENALTIES can be set here. These control the cost of opening up every new gap and the cost of every item in a gap. Increasing the gap opening penalty will make gaps less frequent. Increasing the gap extension penalty will make gaps shorter. Terminal gaps are not penalised."
http://www.clustal.org/download/clustalx_help.html
Basically, a lower Gap Opening and a larger Gap Extension Penalty leads to more an smaller gaps.
In addition, the Delay Divergent Sequences" moves the poorly aligning sequences to the end of the alignment.
"The DELAY DIVERGENT SEQUENCES switch delays the alignment of the most distantly related sequences until after the most closely related sequences have been aligned. The setting shows the percent identity level required to delay the addition of a sequence; sequences that are less identical than this level to any other sequences will be aligned later."
Dear Kate,
I would be careful with excluding sequences with large gaps from your alignment. Although it appears from your screenshot that also putatively conserved regions are missing in the alignment - what points towards the sequences being not closely enough related - you might miss relatives that share the same function but evolved differently in different organisms. Typical examples are loop regions being sometimes signifcantly larger for some memebers of a protein family - e.g. to permit interaction with a binding partner that also structurally differs in that organism, or - the extreme case - whole domains inserted inbetween domains or even into the sequence of another domain.
Basically, such "penalty" depends on how closely releated you want the sequences to be. Are you looking for strictly conserved proteins or are you trying to find similar pathways (signaling or metabolic) with your search?
Apart from that, the major "rule" for looking at such alignments remains - if possible - the identification of conserved regions of (functional) importance. Either being strictly conserved residues with enzymatic function or "motifs" for e.g. protein-protein interactions or substrate binding. Once you have identified such features, for example based on structural or biochemical data available for one or more of the candidate proteins, you can specifically select sequences that share these properties.
Best regards
Johann
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