In a static homology framework, molecular sequences were aligned using their secondary structure as guide, whereby regions of ambiguous alignment were discarded, and pre-aligned sequences analyzed under parsimony and different mixed models in a Bayesian inference.
Sequences were aligned [ 36] and regions of ambiguous alignment were removed when necessary.
First, each gene-set was separately aligned using muscle 3.6 [ 33] and ambiguous alignment regions were removed using Gblocks (version 0.91b) [ 34] with no gaps allowed.
The final aligned matrix (after exclusion of regions of ambiguous alignment, RAAs) contained 4503 nt positions; of them, 1240 were constant, and 2421 were parsimony informative.
Regions inferred to be ambiguously aligned were classified into regions of expansion and contraction (REC), non-pairing regions of ambiguous alignment (RAA) and regions of slipped-strand compensation (RSC) [ 99].
The final matrix included 10,672 bp of aligned sequence data of which 1,902 bp were excluded due to ambiguous alignment (Additional files 6, 7, 8).
Extensive regions of ambiguous alignment mostly corresponding to known expansion regions remained in 18S and 28S after alignment.
Each subject alignment was carefully examined for regions of highly variable, ambiguous alignment, in which a preponderance of indels and repeats occurred, making a probable alignment difficult.
The Hemichordate S. kowalevskii shares with them position −4, and potentially its specific position 6b which could be misplaced due to ambiguous alignment.
We chose to present BLAST-based comparisons, because they tend to mitigate possible over-estimation of OTU-based clusters that can result from sequencing errors, ambiguous alignment, and/or intra-species chimeras [42], [43].
Subject-specific patterns differed between PL and BM, most often in regions of extreme variability and ambiguous alignment; however, no consistent pattern in AA variability across individuals was identified.
