(F ) Computational analysis of CHART-seq peak set and Dali showed that DNA sequences under peaks are not more complementary to Dali sequence than control flanking regions, as judged by either length of aligned regions (left) or alignment quality score (right).
Therefore, these residues were truncated, leaving 545 alignment positions for analysis.
This leaves 167 alignments for which the strand prediction of RNAstrand is compared to the strand prediction of EvoFold.
We left the alignment files of other monkeys in the pedigree unchanged, and then applied the same calling procedure as that described above to this revised set of 105 alignment files.
Translated amino acid sequences were assembled in MEGA 4 [ 47] and aligned using ClustalW [ 48], after which the extreme N- and C-termini of the opsin sequences were trimmed, leaving an alignment 343 codons in length.
Ambiguously aligned positions and gaps were excluded from alignment, leaving 805 unambiguously aligned positions; the alignment is available from the authors upon request.
Ambiguously aligned sites were removed from the alignment, leaving 341 amino acids that were used in the phylogenetic analysis.
Non-aligned N-ends were trimmed, leaving the master alignment presented in Figure 4 and Additional file 3. A consensus HyPRP pattern was developed on the basis of this alignment and verified by MyHits pattern search of the Arabidopsis GenBank proteins at the MyHits website [ 44, 45], where it retrieved the majority of the previously known HyPRP genes.
Sequences that introduced gaps in other sequences were deleted, as were sequences that contained gaps that were not shared by other sequences at the 99.5% gap identity level (i.e. pairs of sequences left in the alignment were 99.5% identical in gap pattern over the aligned region).
Although some are visibly nervous, eyes flickering to right and left, checking their alignment, none puts a foot wrong, or wavers from the carefully set port de bras.
