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Predicted HEAT repeats were defined by three criteria: (1) identification by REP in one or more species using no confidence threshold; (2) secondary structure prediction of two helices connected by a short loop; (3) alignment by visual inspection with HEAT repeat consensus sequences from the Pfam database.
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The observed aerial densities were far too sparse for the insects to have maintained the alignments by visual reference to one another [5], and so the orientation patterns must have been due to individual responses to some environmental cue or cues.
The alignment was checked by visual inspection.
Ambiguous sites of the ERC2/erc2 amino acid sequence alignment were removed by visual inspection as well as with trimAl.
In addition, manual alignment was performed by visual homology to construct a Dedicated Comparative Sequence Editor (DCSE) format in order to perform different phylogenetic analyses.
Alignments are deposited in TreeBase (ID S15183) [ 43].Truncated sequences and those sequences with poor identities were removed, gaps and ambiguously sites in the alignment were weeded by visual inspection.
Clusters with bootstrap values greater than 50% were defined as confirmed subgroups, and sequences with lower values added to these subgroups according to their sequence similarity in the alignment as judged by visual inspection.
Other annotation features were predicted as follows: transmembrane domains by TMHMM 2.0c [ 52]; signal peptides by SignalP 3.0b [ 53]; COG similarities and Pfam domain composition by rpsblast [ 24] The 16S rDNA sequences of S. amnii and 31 related organisms from Fusobacteriaceae family were aligned using the ClustalW program [ 54] and the alignments were corrected by visual inspection.
The accuracy of automatic alignments was confirmed by visual inspection.
Alignments were refined by visual inspection.
Selected alignments were assessed by visual inspection to reveal assembly errors, such as mis-oriented or misordered contigs within scaffolds.
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