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Methylated sites were then estimated based on the alignments mentioned above for each segment using SMAP.
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The primers for the candidate genes of D. montana were designed for one to three unique 200-500 bp regions with Primer3 program [ 23] or manually, using information on the gene alignments mentioned above (primer sequences are available from the authors upon request).
To avoid bias toward a specific structure alignment tool, we evaluate threading alignment accuracy using all the reference alignments mentioned above.
All of the simulated and real alignments mentioned in the paper are available for download from [ 58].
The target sequence is then aligned past the invariant cysteine with all the appropriate JH segments, using the pmf-based alignment mentioned above.
The MAFFT-profile alignment option was used for constructing a combined profile alignment from the above mentioned alignments.
The height of the whole femur was calculated after applying the 3 alignment methods mentioned above.
All of the above mentioned alignment methods are fully automated, i.e., they construct alignments following a fixed set of algorithmical rules.
All new putative opsin sequences of A. filiformis were included in phylogenetic analyses based on the previously mentioned alignment.
To further compare individual aligner with these two alignment algorithms mentioned above, we performed evaluation and comparative analysis of these aligners in terms of computational performance, alignment accuracy, and application-specific features.
More specifically, we repeated each alignment in the previously mentioned datasets by using (i) the normal procedure, enabling the program to build its own guide-tree, (ii) forcing the use of the correct tree (or a canonical species tree) as a guide-tree and (iii) forcing the use as a guide-tree of a synthetic 'wrong' tree having the maximum split-distance to the correct tree.
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