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Manual examination of alignments showed that such indels occur not only as extensions to pre-existing structures, but are introduced in existing domains into the middle of the structure.
Sequences with 10% or more undetermined bases or chimeric features, as determined by screening with MALLARD [10] followed by manual examination of alignments generated by BLAST against GenBank, were removed.
Examination of alignments of the full-length proteins revealed 10 fully conserved residues (R112, G129, N131, R147, G151, E153, N164, Y166, G211, D213 where the numbers refer to the alignment positions in Fig. S2); all were located within the β-domain.
This was checked by manual examination of alignments.
Closer examination of alignments using reference sequences from the IMGT/HLA database and the IGV browser showed that the MIT-NGS calls were correct rather than the original SBT results.
These 10 protein coding gene regions were aligned by eye to the dataset used in Near et al. [ 40] and confirmed by examination of alignments of the inferred amino acid sequences.
Similar(53)
Intriguingly, examination of alignment data for the cysteine-rich metal-binding domain alone reveals that in a subset of species, mainly protozoa, sequences with a corresponding pattern of cysteines are found as tandem repeats, and lacking the helical domain.
However, more detailed examination of those alignments and trees disclosed problems in the alignments for all of the candidate novel sequences.
An examination of the alignments with various parameters highlighted the increase in quality of alignment in the later versions of PASS2.3 (2008), which will help in linking representative superfamily members to cover rapidly expanding sequence space.
Manual evaluation of positively selected genes involved examination of the alignments, where in families with branches with Ka/Ks>1 and alignments where some sequences had a high percentage of gaps, the effect of these sequences on the alignment and subsequent calculation of Ka/Ks ratios was evaluated.
Examination of resulting alignments and subgroup specific dendrogram construction (UPGMA, BLOSUM62) were performed using the Jalview alignment editor [ 70].
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com