2.6. Annotation of Non-Coding RNAs

Annotated ncRNA candidates from the first assembly of D. vexillum were mapped in the new assembly as described in Section S6. At the same time, homology blastn and HMM strategies with their corresponding metazoan-specific CMs and default CMs evaluation have been applied following the methodology proposed in [26], to annotate candidates that have not been detected with the mapping strategy. The tRNAs genes were found using tRNAscan-SE v.2.0.3 with default parameters. A final check of candidates was performed to ensure that reported Rfam families contain at least one Metazoan sequence in their original seed alignment. These last step was performed to report possible false-positive families that could be retrieved applying the default Rfam models directly to the genome.

In order to annotate the position of mature sequences from miRNA candidates, MIRfix [58] was used. The miRBase (v.22) mature and hairpin sequences were used as initial sequence resource. Via RNAcentral database [59], the cross-link between miRBase and Rfam (v.14.1) was retrieved, and a list of Rfam families were classified as annotated in both databases. In this case, MIRFix corrected the mature position within the corresponding hairpin sequence. After that, the remaining seed sequences that have not been annotated in miRBase were included to be evaluated by the same methodology, but with the mature family-specific sequences. Final correction and annotation of those families, allowed the re-build of multiple sequence and structural alignments from the Rfam defined sequences, as a stockholm alignment. Given those results, the D. vexillum miRNA sequences annotated in this study, were processed as subject to annotate their mature sequences, based on previously detected matures in the Rfam families. At the end, positions of the most probable mature sequences and the corresponding alignments in stockholm format for each miRNA Rfam family were retrieved. Those genome annotations can be assessed via the described genome browser.