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Both alignments were tested for recombination with the Single Breakpoint Recombination SBPP) tool [87] in the DATAMONKEY web-server [88]; evidence for recombination, inferred by the small sample AIC score, was only found for HIV-1 Control dataset.
Prior to performing selection tests, alignments were tested for recombination using Genetic Algorithms for Recombination Detection analysis (GARD) implemented in Datamonkey.
To minimize the influence of recombination in the PS scan, the alignments were tested for intragenic recombination using GARD (Kosakovsky Pond et al. 2006).
The resulting alignments were tested for positive selection by applying test 2. The log-likelihood difference (2Δ L) of these tests was recorded.
The rRNA and RPB1 alignments were tested for saturation using the methods of Steel et al. [ 74] and Xia et al. [ 75], implemented in DAMBE.
The quality of the COI/COII and haemocyanin alignments were tested using GBlocks 0.91b [ 81, 82] tolerating gap positions within final blocks, which retained between 98-100 % of the original alignment.
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To avoid cross-hybridization, all other alignments are tested using the specificity filtering test.
This combined approach, which is applicable to extremely long alignments, is tested with simulations.
The presence of saturation in base substitution for each of the alignments was tested using DAMBE5 [ 20].
The specificity and coverage of the resultant alignments are tested against the orthologous groups defined in the eggNOG database v3.0 [ 42].
The seed alignments are tested and improved by manual curation, and by application to large databases like the Universal Protein (UniProt) database [ 13].
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