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The third issue is mapping, which with short tags (around 25 to 35 bp) can be ambiguous in regions of high homology or in repeat regions.
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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.
Regions inferred to be ambiguously aligned were classified following Gillespie [ 49] in regions of expansion and contraction (REC), non-pairing regions of ambiguous alignment (RAA) and regions of slipped-strand compensation (RSC).
Alignment of Th. intermedium sequences and other Triticeae was straightforward in all exon regions and a major part of intron 9, but ambiguous in two regions.
There were a few ambiguous cases in variable regions of the protein, where the alignment was uncertain.
Ambiguous regions of the alignment were removed using Gblocks [ 53].
Priming sites and ambiguous regions of the alignment were excluded.
The data was further edited by eye and exported from ARB [ 25] using an 'in-house' filter to remove highly ambiguous regions of the alignment.
Subsequently, ambiguous regions of the protein sequence alignments were filtered using TrimAl v1.4rev7 (Capella-Gutiérrez et al. 2009) under the parameters set in the automated1 option.
The ambiguous regions of alignment were discarded and eventually 720 nucleotide bases were obtained.
Although a comparison of the two versions is beyond the scope of this study, a preliminary analysis indicates very few differences between the two, mostly found in an ambiguous region of our sequence.
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