Stop coordinates were adjusted only if there were predicted selenocysteine codons, frameshifts, or nonsense codons interrupting an alignment of protein sequences.
Only proteins containing the complete module R-B1-B2-CC were aligned in a first step, eliminating from each sequence the portion downstream of the coiled-coil domain and all segments that caused a gap to interrupt the alignment.
Genes missing from A. carolinensis annotations can be attributed to gaps in the Anocar2.0 assembly; misassembly in genome scaffolds would interrupt contiguous alignments of transcripts at contig sequence boundaries.
These data were included as a low BlastN score does not invalidate them because some oligos predictably span introns (which would interrupt BlastN alignments) and the cutoff score used is potentially above the actual hybridization threshold which is influenced by the abundance of the target messenger RNA, the abundance of competing non-target RNAs, and the Tm.
The alignment was interrupted by an insertion of 444 bp in the E. grandis sequence and the identity level in the remaining 5' sequence dropped to 85%.
Sequences longer than five amino acids were also included, even if the alignment was interrupted by one or two nonmatching amino acids.
Exonerate (Slater and Birney 2005) was used with the parameters "exhaustive" (which turns all heuristics off and allows for the most sensitive search), "percent10" (which sets the required sequence identity threshold for putative hits to 10%), and "est2genome" (which models cDNA to genome alignments and allows for alignments to be interrupted by nonconserved regions such as introns).
With the fast mode option, this threshold is increased by 0.25 which has the effect that the extension of fragments during the pairwise alignment phase is interrupted far more often than by default.
This was accomplished by summing bitscores for otherwise continuous alignments that were interrupted by gaps of 10,000 or fewer bases.
The annotations of repetitive regions were downloaded from the UCSC genome browser, including transposons, microsatellites, simple tandem repeats, interrupted repeats, segmental duplications, self-alignment regions with similarity score > 80, and other repeats masked by RepeatMasker (http://www.repeatmasker.org).org
