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Primer sequences were deleted and the initial alignment was further modified with GBLOCKS [ 75] to remove ambiguously aligned internal positions and to trim the alignment at the 5' and 3' ends.
To construct the dispersion ratio from a pair of aligned protein-coding sequences, we begin by identifying all positions j in the alignment at which the amino acids disagree.
There is occurrence of Inorganic Silt (MH and ML) and OH in four locations along the alignment, at chainages 26.85 km, 27.25 km, 34.75 km, and 35 km.
It is likely that the removal of gapped positions in the alignment at least partially contributes to the observed lack of clustering in the histamine receptor family, in particular as the histamine H1 receptor crystal structure was also used to select the binding site residues (see Methods).
Furthermore, NSP2 (for this gene, we sequenced 33 strains in total) was used to examine the alignment at the amino acid level [18], [24], [46].
Amino acid conservation at every position of the peptide sequence alignment was plotted using the "sequence logo" method [79], by pasting the alignment at the logo website (http://weblogo.berkeley.edu/logo.cgi, [80]).
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The catalytic domains were aligned using Clustal X [38] and the alignments at the catalytic centre of the catalytic domain was used to derive the search motifs [12].
The alignments at candidate positions were visually inspected using the Integrative genomics viewer (IGV) [ 34].
We then separated the alignments at the detected breakpoints within the region, estimated MP phylogenetic trees (1000 bootstrap replicates) for the individual sections, and checked for incongruent topologies.
Interaction matrices were generated from the alignments at a resolution of the restriction fragments and at 40 kb resolution (using BEDtools on a 40 kbp binned genome).
At the gaps in the alignments, at least 10% of the members (or at least 2) had to have non-gap characters in the gap position to be included in the diversity calculation of the alignment.
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