The intact OR sequences were codon-aligned using MAFFT, the alignment was manually edited and alignment gaps present in >85% of the sequences were removed.
All sequence chromatograms were controlled manually and sequence alignments established in BioEdit [ 41] by manual adjustments.
Gaps were removed manually, and all alignments were analyzed using RAxML v 7.2.6 [ 86] using the Gamma model of rate heterogeneity and the WAG substitution matrix with 100 bootstrap replicates.
The sequences were edited manually (BioEdit V.5.0.9) and alignment, phylogenetic and molecular analyses were performed using MEGA version 4 with neighbor-joining method under Kimura's two parameter.
The 22 tRNA genes were also aligned manually, and ambiguous and gap alignment were excluded from the phylogenetic analyses.
As alignment errors can create false positives in the detection of positive selection footprints, each cluster suggested to be under positive selection was again checked both automatically – using muscle [ 72] and trimAL [ 73] for creating and cleaning alignments (muscle-trimAL method; see Additional file 1) – and manually for alignment errors.
The sequences were manually aligned and the alignments were concatenated.
For the U1A70kF-RNA complex, an initial molecular replacement solution was found in Phaser (McCoy et al., 2007) using part of the structure of U1-A complex with SL2 RNA (Oubridge et al., 1994) and a homology model of the U1-70k RRM, which was built with Modeller (Mart-Renom et al., 2000) using multiple templates and manually refined alignment as an input.
Following automated alignment, it is usually necessary to check, correct and trim the alignment manually, and check sequence differences between individual sequences, which is easily done in the sequence editor included in ProSeq3.
In a few cases, e.g., for some molecular features with high fold change and high significance, integration and alignment were manually confirmed and were in good agreement with XCMS results.
