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The combined data matrix included 1974 aligned positions (ITS: 864 bp; rpL16: 1110 bp), of which 29% of the aligned sequences (31.6% in ITS and 26.9% in rpL 16) were excluded because of uncertain alignment.
Hence we did not remove areas of uncertain alignment, especially since the AU plots of both the Solenopsis and Apis BAli-Phy alignments suggest that there are still high quality alignment blocks within these "problematic" areas to warrant their inclusion in the overall alignment procedure (Figure 1b).
Close inspection of protein alignments revealed that the rhesus monkey CCT8L sequence included an anomalously diverged segment of about 50 amino acids of uncertain alignment.
Initial sequence alignments were generated using MUSCLE v3.8.31 (Edgar 2004), with some adjustments made by eye and regions of uncertain alignment excluded using Se-Al 2.0 (Rambaut 2008).
Regions of uncertain alignment from all three genes, coxI intron sequences [ 28], and known RNA editing sites for atp1 and coxI were excluded prior to analysis [ 46].
Expression levels for SSOGs were quantified with these maps using Cufflinks (Trapnell et al. 2010), correcting for fragment bias (" –– frag - bias - correct") and uncertain alignment location (" –– multi-read-correct") and using default parameter values otherwise.
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This sequence alignment falls on the empirical threshold which distinguishes instances of significant similarity from uncertain alignments [41].
Sequences with highly divergent regions or gaps resulting in uncertain alignments were excluded from the further analysis.
Incomplete sequences, and highly divergent regions or gaps resulting in uncertain alignments were excluded from the analysis.
Incomplete sequences, and highly divergent regions or gaps resulting in uncertain alignments were excluded from the further analysis.
Mismatches were mainly from polynucleotide artifacts from the 454 sequencer and uncertain alignments were examined manually to confirm that they were not editing sites.
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