Reads remaining unaligned at this point were aligned with LAST [ 9]. Results were combined into a single alignment file in SAM format and reported versus single reference (the Flybase v5 D.mel reference [ 19]).
Resulting alignments were then concatenated in a single alignment of 46,607 residues, in which missing proteins in some orthogroup alignments were replaced by tracks of gaps.
The alignment stage extends matching regions as far as possible, merges overlapping matches, links matches that fall in order on the genome into a single alignment, and fills in regions of the alignment corresponding to gaps of identical length in the query and genome sequences.
The three recurrent regions, referred to as A1, A2 and A3, respectively, were merged in silico in a single alignment file, termed As, to increase the resolution and improve the capacity to define the evolutionary trajectory of the recurrence specimen.
For each reference alignment, a "combined score" was calculated corresponding to the maximal score possible if all correctly aligned blocks from each program were combined in a single alignment.
After cleaning up the nucleotide alignments for poorly aligned regions, the 1,486 multiple alignments were concatenated in a single alignment of 431,655 codons.
Visual depictions of the alignment as in the image at right illustrate mutation events such as point mutations (single amino acid or nucleotide changes) that appear as differing characters in a single alignment column, and insertion or deletion mutations (indels or gaps) that appear as hyphens in one or more of the sequences in the alignment.
As the pits in a single alignment on both sides of the indentation show equivalent asymmetry, they must represent the surface intersection of asymmetrically shaped dislocation loops.
We reiterated this up to three times per gene, generating up to three additional LGT events in a single alignment.
The poorly aligned positions and divergent regions in each alignment were eliminated with GBlocks program [ 42] and the resulting blocks were concatenated in a single alignment of 15195 positions.
