Structure of the iBeetle-Base
The iBeetle-Base stores gene related information for all genes of the official gene set (red box). Among others, RNA and protein sequences can be downloaded and links lead to the respective annotation in the genome browser. Further, the Drosophila orthologs are displayed including links to FlyBase. Wherever available, the phenotypic data gathered in the iBeetle screen is displayed below the gene information in separate sections for the pupal and larval screening parts (yellow box).
Searching for gene IDs or phenotypes
The information can be searched from two sides: First, the “Gene search” uses gene IDs (TC number) or name of the ortholog or iB number to display the details page (gene search, left blue box). There, all information available for this gene is shown (blue and yellow boxes). If the entered search term is equivocal, a results list presents all hits for selection by the user.
Second, the “search for morphological defects” allows searching for affected structures and/or the nature of the defects. Here, the result list presents both the annotation and the pictures of the searched entity for further selection. Links to the respective details pages gives all information available for this gene.
Note that for all genes of the official gene set the gene information is s tored at iBeetle-Base while only a subset of the genes has been screened in the iBeetle screen.
iBeetle is a first pass screen
The iBeetle Screen is a first pass screen, i.e. the experiments were performed only once and off target controls were not included. Further, we aimed at keeping false negative results as low as possible with the trade-off of elevated false positive annotations. Finally, we deliberately used an intermediate dsRNA concentration (1ug/ul) in order to reveal the phenotypic series. Hence, the data presented here needs to be confirmed with the original fragment and non-overlapping fragments of dsRNA before being publishable.
Highly penetrant phenotypes with a direct phenotype-genotype relationship (e.g. lethality, wing blistering) were reproducible at a close to 100% rate in our hands. Regarding processes where the relation between gene function and phenotype is complex (e.g. embryonic development), the rate of reproducibility was between 70% and 80%. Reproducibility of low penetrance phenotypes (<50%) was significantly lower.
The search at iBeetle-Base allows for very specific searches (e.g. “larva, leg, size decreased”). However, in order not to miss interesting phenotypes, we recommend initiating your search broadly for defects of the structure and stage you are interested in (larva, leg). Further, a penetrance of >50% is recommended in order to increase the portion of reproducible datasets. Next, the list should be consolidated by browsing the pictures and detailed annotations displayed in the search results. Interesting results can be marked by checkboxes. Finally, the respective details pages should be browsed in order to learn more about additional phenotypes, the gene structure and sequences and its orthologs.
Rescreen strategy for the identification of morphological defects
Phenotypes need to be confirmed by both the original dsRNA fragment (see sequence on the details page) and a non-overlapping fragment for off target control. A higher concentration (e.g. 3ug/ul) than the one used in the screen (1ug/ul) is recommended because in some cases, this will reveal s tronger phenotypes. For mid scale re-screening it may be advantageous to buy dsRNAs commercially from Eupheria.com.