Background information
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.
Annotations - Terms
Most phenotypes gathered in the iBeetle-Screen were annotated in a machine-readable way utilizing the EQM system. This includes the morphological Entity that was affected (such as leg), the Quality of the phenotype (such as size) and a Modifier that further describes the phenotype quality - for instance: "leg size decreased". The terms were chosen from controlled vocabularies.
The annotation list is downloadable under Resources page. See Schmitt-Engel et al. (2015) or Dönitz et al. (2015) for more details.
Reproducibility
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.
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.
Screening procedure
Pupal injection procedure
Day 0
10 female pupae of the pBA19 strain (muscle enhancer trap line) were injected with dsRNA.
3 days post injection (dpi)
Hatch control: Pupal and adult lethality as well as metamorphosis defects (molting, eclosion) were documented. For mating, 4 males of the black strain were added.
9 dpi
First egg-lay was collected and incubated for cuticle analysis. Adult lethality and egg production (reduced/ no egg-lay) was documented.
11 dpi
Second egg-lay was collected and incubated for embryonic muscle analysis. Adult lethality and egg production (reduced/ no egg-lay) were documented.
13 dpi: Egg productivity and Ovary analysis
The percentage of hatched larvae was documented and not hatched larvae/ eggs were embedded for cuticle analysis (15 dpi). In case of a reduction of egg production, 4 injected females were dissected to analyze the gross morphology of the ovaries.
14 dpi: Analysis of embryonic musculature and early embryonic development
Offspring of the injected females (hatched and not hatched larvae/ eggs) were analysed for embryonic lethality and muscle defects.
15 dpi: Analysis of larval instar 1 cuticle
Offspring of injected females were analysed and cuticle phenotypes were annotated.
22 dpi: Stink gland analysis
Documentation of defects in abdominal and thoracic stink glands (colour, size, content) of the injected femals.
Larval injection procedure
Day 0
10 Instar larvae (L6; Female larvae were derived from a cross between D17Xred and pearl strainsD17Xred and pearl) were injected with dsRNA 1 μg μl-1
11 dpi
Analysis of pupal Morphology, Larval lethality and muscle defects
23 dpi: Adult Morphology and fertility
The percentage of hatched larvae was documented. In case of a reduction of egg production, 4 injected females were dissected to analyze the gross morphology of the ovaries.
6 weeks post injection: Stink Gland Analysis
Documentation of defects in abdominal and thoracic stink glands (colour, size, content) of the injected females.