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Clade model analyses revealed that, after duplication, the selective regime experienced by many alignment sites changed from weak purifying selection to either neutral evolution or weak positive selection.
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Many alignment algorithms exist.
There are many alignment formats available.
Many alignment methods often produce reasonable alignments which score worse than alignments with alternative templates.
Many alignments did not involve active site residues or residues playing other structural and functional roles.
The presence of truncated sequences in many alignments meant that the exclusion of gap sites sometimes ruled out the entire alignment (i.e., when all alignment columns had at least one gap).
This approach appears particularly beneficial when large Markov rate matrices are used, for example in the calculation of codon substitution models, or when treating alignments with many sites (as opposed to many taxa).
Third, we identified multiple copy sequence segments with alignments to many sites (≥ 10).
Conserved domain III, which has been shown to function in transactivation in rat FoxA2 [ 36] contained many ambiguous sites in the FoxA alignment (see Additional file 3(A)) due to sequences from the Protostomia lineage and variations in selection pressure were observed in the four sites, through site analysis, that did contain amino acids from these species.
Multiple alignments were then performed using MUSCLE [ 66], which automatically removed ambiguously aligned sites or sequences with too many gaps.
One might expect higher conservation in the core residues and thus a greater distinction between site and non-site scores; however, many fewer alignment positions exist in the cores than the whole sites, and in some cases the increased statistical power gained by considering the whole site meant that we obtained a significant result using the whole sites but not the cores.
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