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While this reduced data set lacked power due to the small number of species and slight divergence between them, the primate-only data set has the most confident alignment.
Thus, the tests require (i) well-characterized transcription factor binding specificity and (ii) confident alignment of a binding site to a single specificity matrix.
To focus on core genome regions with confident alignment and homology assessment, we retained only the 1,351 core genome blocks that were at least 1 kb in length (Dettman et al. 2013).
Accordingly, higher values of Qs represents a more confident alignment, and the mapping quality score Qs will be lower or zero for reads that could be mapped to multiple locations with nearly the same number of mismatches.
The portion of animal Nanos proteins with conservation sufficient for confident alignment (48 amino acids) is too short to yield significant phylogenetic signal (supplementary material Fig. S2C, note low support values), but Oncopeltus Nanos does contain the diagnostic 2× CCHC) zinc finger domain found in all Nanos orthologs (supplementary material Fig. S2D).
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Unique best hits were filtered to confident alignments with posterior probability ≥ 0.9.
Table 2 shows that almost all confident mappings from MAQ and BWA exist in consistent pairs although MAQ gives fewer confident alignments.
We also eliminated 14 sequence alignments from analyses either because extensive indels made confident alignments impossible or because a substantial portion of the A. lyrata sequence did not appear to be homologous to the A. thaliana sequence.
Searches were initially run with a stringent e-value threshold of 1e-35 until convergence (to obtain confident alignments and more robust profiles) and later the threshold was relaxed to the value corresponding to 0.01 times the score of the first false positive and searches were continued until convergence.
In addition to requiring a set of three genomes for each group, our analyses also demanded that their divergence levels be within a fairly narrow range, low enough to allow confident alignments of noncoding regions, yet sufficiently high to allow for the accumulation of indels.
Because of difficulty in aligning the 4.67-kb noncoding region between ADH4 and ZRT1, we used only ∼200 bases downstream of ADH4 and 800 bases upstream of ZRT1, where we were confident of alignment.
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