A total of 456 complete amino acid sequences (452 available in databases and 4 newly predicted) can be reliably aligned, and the alignment of representative sequences from each of the 5 subfamilies is shown in Figure 1.
Regions of the optimized alignment which could not be reliably aligned were eliminated from analysis (data alignment available upon request), resulting in an alignment of 525 bp for 16S, 520 bp for 12S and 1,149 bp for the Cytb dataset, for a total of 2,194 bp.
Application of Gblocks [ 65] at default settings lead to a final alignment of 2572 reliably aligned amino acids.
To generate a multiple sequence alignment that consisted of only reliably aligned amino acids, thereby minimizing possible artifacts introduced due to alignment error, we used the online web server GUIDANCE [ 52].
Reliably aligned regions in full length alignments were selected with Gblocks [57].
Multiple alignments with Probalign retained 491 reliably aligned genes from a set of the 497 orthologous genes.
Finally, we discarded sequence sections from the alignment that could not be reliably aligned across all taxa, because they seemingly have diverged sufficiently to preclude meaningful analysis.
All invertebrate-type lysozymes could be reliably aligned to each other at both protein (alignment 2; Fig. 3) and DNA sequence level (alignment 3; position of indels is identical between the two alignments).
For each reference alignment, blocks are defined that correspond to the reliably aligned regions, using the RASCAL [60] program.
After that, BWA clears the BWT index from the memory, loads the 2 bit encoded reference sequence and performs Smith Waterman alignment for unmapped reads whose mates can be reliably aligned.
While both these regions contained small indel events and multiple nucleotide degenerate sites, resulting in multiple plausible evolutionary histories, the posterior alignment distribution from BigFoot exhibited how our joint model reliably aligned all instances of the binding site together.
