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By performing three independent alignments using the staggered genomic indices and then superimposing the resulting alignments to show all aligning sequences, the enhanced-BLAT (eBLAT) detects as much as 75% more conserved sequences when evolutionary distant sequences are aligned [22].
These decreased numbers showed that the screen was not sensitive to the terminator filter, but instead depended solely on the cut-off value, as more conserved sequences could have more co-localized terminators.
We used all ranks of the NCBI taxonomy, placing more conserved sequences higher up in the taxonomy (i.e., closer to the root) and more distinct sequence onto nodes that are more specific (i.e., closer to the leaves, which represent species and strains).
As demonstrated in Figure 1, the pairwise eBLAT alignment exhibited only a modest increase in the identification of shared sequences between closely related species over the conventional BLAT alignment; however, eBLAT identified significantly more conserved sequences when the D. melanogaster genomic fragments were aligned to the more evolutionarily distant orthologs.
Moreover, the original binding peaks with an average phastCons score >0.32 even tended to be less conserved than randomly selected NCRs, and the opposite was true for the putative CRMs, indicating that the predicted CRMs contains more conserved sequences than do the original binding peaks.
On the other hand, these ancient loci have to be detectable across more lineages in order to be included in the older groupings and it is possible that these higher levels of conservation are partly due to an overrepresentation in these groups of more conserved sequences that can be robustly aligned across such a wide phylogenetic range.
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The observation that more highly conserved sequences are less polymorphic is consistent with the hypothesis that evolutionary conservation highlights functionally important sequences.
Alternatively, these extraordinarily long UCRs might interact with RNAs or DNAs in other genomic regions and such interactions might require more highly conserved sequences.
PAML deletes sites in the ancestral sequence where any one sequence contains a gap in the MSA, whereas FastML uses a binary matrix to reconstruct indels and adds them back into regions of more highly conserved sequence.
By contrast, the right peaks for single putative CREs in CDRs and the randomly selected short k-mers are much larger than their own left peaks, indicating that there are much more conserved short sequences than non-conserved ones in CDRs, which is expected as most CDRs are highly conserved.
In contrast, site 317 was more conserved in sequences from vaccine recipients — in 95% of vaccinees compared to 71% of placebo recipients.
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