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Protein sequence alignments were generated with CLUSTALW (version 1.8) [47] at NPS@ [135] with manual alignment of some positions.
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The extreme branch length differences between Gnetales and other lineages for sites at the most varied end of the OV-sorted alignment suggests an issue with alignment of some amino acid positions, despite a conservative approach being used in generating the sequence alignments in the present study.
Alignment of landmark cone positions to reference positions was accomplished with two linear transformations: scaling (shrinking and expanding) in slow scan direction and shearing in fast scan direction.
After the removal of ambiguously aligned positions and portions that have not been sequenced in Convoluta, an alignment of 11,959 positions was obtained.
However, views of the NextGene alignments revealed that some positions showed mixtures of the same false positive substitution or deletion recorded by the variant caller.
For the second approach, SATé returned a best-scored ITS alignment of 1,296 positions for 300 taxa (Alignment S2).
In summary, our large alignment of 11,959 positions strongly rejects the grouping of acoels with platyhelminths, and more generally protostomes.
We further removed all positions for which Convoluta was not present (due to partial gene sequences), resulting in a final alignment of 11,959 positions.
The concatenation of ribosomal proteins yielded an alignment of 2362 positions after removal of ambiguous sites.
A second alignment of 9,745 positions was generated by removing potential compositional bias with BMGE [ 36].
However, variation is overall lower than engrailed with only 27 variable sites in the nucleotide alignment of 333 positions.
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