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Using ad hoc scripts and manual selection, multiple sequences in each species were trimmed, retaining the best-annotated sequences in those alignments.
Each sequence was annotated using the BLAST alignment with the lowest expected value (≤ 1 × 10-6) in those alignments where the reading frame was in the positive orientation.
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We excluded from our analysis those alignments in which there was at least one site without known solvent accessibility.
To eliminate erroneous and nonorthologous gene alignments, those alignments in which pairwise dS and/or dN exceeded a pre-specified threshold between any pair of species were excluded from analysis.
CrossMatch aligned two regions, but contig sequence fragments in those two alignments overlap by 315 bases and additional analysis is required for breakpoints identification.
In addition, the mapping directions were also observed in those hybrid alignments; for example 2014draft G448 was found to reversely aligned to M129 (Fig. 6a).
There are only 68 archaebacteria in our genome sample, and it is evident in figure 2 that among those alignments and trees where eukaryotic genes branch with archaebacteria as sisters, the eubacteria are underrepresented.
Trimming of those alignments ended in 8,031 final alignments with an average length of 402 bp.
In addition, to filter out those alignments that lie within intronic intervals implied by the spliced alignments, we set the pre-mrna-fraction as 0.25.
In general we considered only those alignments where the query sequence (small RNA) was mapped to the database sequence (tRNA) along 100% of its length.
Several subfamily alignments are given in Additional File 1 and the seed sequences are those explicitly shown in the alignments.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com