RNAz was applied to the filtered input alignment slices in both reading directions.
In a series of filtering steps, sequences were removed from the individual alignments or alignment slices if they are (a) shorter than 50 nt, or (b) contain more than 25% gap characters, or (c) have a base composition outside the definition range of RNAz (e.g. GC content > 0.75 or < 0.25).
For alignment slices with more than 6 sequences, rnazWindows.pl selects a representative subset consisting of the D. melanogaster sequence and five additional sequences in such a way that 6 sequences are as evenly distributed in the dataset as possible and approach an average pairwise sequence identity of 80%, the optimal working range of the RNAz program.
Alignment of slices – For uploading the slices into Amira, consistent physical dimensions of the pictures are needed for registration.
The alignment was sliced into windows of constant size and fixed overlap and a 100-replicate maximum-likelihood (using HRV-93 as an outgroup) was computed for each window.
Preprocessing of functional data consisted of correction for head motion (trilinear/sinc interpolation, the first volume of the first run functioned as reference volume for alignment), interscan slice-time (sinc interpolation), linear drifts removal, and temporal high-pass filtering (threshold at 7 cycles per run).
The alignments were sliced into 120 long blocks with a step size of 20 and only alignments with more than 65 columns were reported.
Pre-processing involved temporal alignment of volume slices (to correct for asynchronous slice acquisition), normalization within each scanning run to a fixed image intensity value, and then correction for motion using a rigid-body rotation and translation algorithm [19], [20].
Biplanar slice alignment was used.
Although these datasets can be used to generate highly resolved digitized 3D models of invertebrates as well (see, for example, [ 47, 48]), these analyses are extremely demanding in terms of specimen preparation, slice alignment and 3D reconstruction.
