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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).
Legend: Structure of the iBeetle-Base. The details pages display both gene information (red box) and phenotype information (yellow box). It can be searched by the “Gene search” for gene names and IDs and for iB numbers. the “search for morphological defects” allows searching phenotype annotations. Note that gene information is available for all genes while phenotypes are available only for a subset.

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.

Legend: In the search for morphological defects choose the developmental stage that you are interested in (larva, pupa, adult). Enter the morphological structure that should be affected by typing into the field and selecting from the terms that are displayed (a). Alternatively, browse the morphological terms by clicking (b). It is advisable to choose a penetrance of >50%. The number of datasets matching the criteria is displayed (c). You may add terms to specify the nature of the defect and you may combine searches by logical operators. See text for further hints on the search strategy.
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.

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.

Search strategy

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.
Screening procedure and terms

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.

Note: The adult morphology was not analyzed systematically at 3/9/11 dpi. Only obvious phenotypes, visible without magnification, have been annotated.

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.

Terms used in the pupal injection procedure

larval pantagmatic defects
At least two tagmata (head, thorax, abdomen or terminus) show similar/ comparable defects.
empty eggs
Just empty shells are visible, no cuticle has been developed.
strong defects
L1 cuticle larvae which show severe defects. These strong defects are subdivided into three categories
strongly fragmented (cuticle crumbs)
No segments or appendages are identifiable, only residues of cuticle and bristles can be identified.
cuticle remnants (with unidentifiable segments)
Segments or parts of appendages are recognizable, but identity is not clear.
cuticle remnants (with some identifiable segments)
At least one segment or appendage is clearly regonizable.
number of eggs on slide
affected embryos usually (not always) do not hatch. Only the not hatched were used for cuticle analysis.
For the embryonic muscle analysis the hatched as well as the not hatched were analysed.
total number of affected eggs/embryos/larvae
Summary of all different phenotypes/ phenotypic series which were annotated.
inside-out
The L1 larva shows a partial or complete inversion., e.g. bristles, appendages or parts of the abdominal segments are inverted into the interior of the cuticle.
starved
Dissected females show a strong resorption of fatbody predominately in the abdomen and the oogenesis is blocked. These are considered as eventually lethal phenotypes and the blocked oogenesis probably a secondary defect due to starvation.
eclosion not fulfilled
The emergence of the adult from the pupa stage is interrupted. This phenotype shows pupal as well as adult features.
Please see the help page for more information

Larval injection procedure

Day 0: 10 female L5-6 larvae of the D17Xhom strain (females express RFP in the eyes and in the CNS; pupae express GFP in the thorax) were injected with dsRNA.

11 days post injection (dpi): Pupal morphology Documentation of larval/ pupal death and defects of metamorphosis (eclosion, delayed development). Screening for alterations of pupal head and appendages, pupal thorax, pupal thoracic musculature, pupal legs, pupal wings, pupal abdomen and pupal genital lobes.

16 dpi: Adult morphology Documentation of larval/ pupal and adult death and defects of metamorphosis (eclosion, delayed development). Screening for alterations of adult head and appendages, adult thorax, adult elytra and hindwings, adult legs and cuticle properties. For mating 4 males of the black strain were added.

19 dpi: Sieving and egg-lay Documentation of adult lethality and enduring metamorphosis defects.

22 dpi: Ovary analysis (Fertility) In case of a reduction of egg production 4 females were dissected and the gross morphology of the ovaries were analysed.

41 dpi: Fertility and stink gland analysis Documentation of defects in abdominal and thoracic stink glands development (colour, size, content) of the injected females. Check for fertility of injected larvae (no or decreased offspring).

Terms used in the larval injection procedure

pupal/ adult pantagmatic defects
At least two tagmata (head, thorax, abdomen or terminus) show similar/ comparable defects.
pupal molt not fulfilled
The hatching from the larva to the pupa is interrupted. Either it dies as prepupa or the phenotype shows larval as well as pupal features.
eclosion not fulfilled
The emergence of the adult from the pupa stage is interrupted. This phenotype shows pupal as well as adult features.
starved
Dissected females show a strong resorption of fatbody predominately in the abdomen and the oogenesis is blocked. These are considered as eventually lethal phenotypes and the blocked oogenesis probably a secondary defect due to starvation.
Morphological search

Choose the stage and click on a term on the list of morphological structures, which will appear upon typing into the respective field.
Optional: Choosing a penetrance of >50% will strongly reduce false positive hits.

Morphological structure

Select the anatomical structure of the phenotype by using the:

  • auto complete feature. After three letters are typed in the search field, a list of matching values is displayed. Please select one from the list.
  • list of available structues by clicking on . The list of available structures are displayed hierarchically . The number in parentheses idicate the number of data sets linked to this term in any developmental stage.

Penetrance

Estimated portion of individuals in the preparation showing the selected search term combination.
Phenotypes with penetrance >50% are reproducible with high frequency.

Optional further specifications

Be careful with choosing further specifications: You may miss important phenotypes because morphological phenotypes can be correctly described with different term combinations.

We recommend to search rather broadly for the morphological structure of your interest and then reduce the number of hits by browsing through the pictures.
Phenotypes with a penetrance of 30% are frequently not reproduced, especially if they come in combination with many additional annotations.

Total penetrance

Estimated portion of individuals showing any phenotype in the preparation.
This is not equal to the sum of all annotated phenotype penetrances because cuticles can show several different aspects that were annotated separately.

Your search

Combine searches by clicking the “AND” “OR” “NOT” buttons.
Your current combination of search terms is displayed. Modify the single aspects by clicking on the respective button, or change the logical combination. Remove aspects using the “Remove” button.

Search results

Filtering and refresh

You may exclude single entries from the search by deselecting the check box at the beginning of the row. The check box in the header of the table (de-) selects all rows on the current page of the table.
With the "Refresh" button you can remove the deselected entries from the result set and refresh the table.

Export search results

You can export your search results as csv, xls or pdf file. If you have removed already some entries, only the remaining are exported.

  • The csv file contains the iBeetle IDs of the search result, one ID in each line
  • The xls file contains the iBeetle IDs and the features of the screening days with the matched features. These are the same features as displayed online. To view all features please adjust the row height of the table to the optimal width.
  • The pdf file contains the iBeetle IDs together the features of the screening days with the matched features.

iBeetle details page

Former TC numbers

If one gene is splitted or genes are merged, the original TC numbers are listed. One listed former TC number indicates the previous gene has be splitted, serveral former TC number represents a merged gene.

Target gene

The sequence from the iB fragment is matched against the genes from the official gene set. The matche(s) is/are visualizied in a genome browser.

Fly ortholog

The information about the homolog genes in Drosophila is provided by OrthoDB http://www.orthodb.org/. The homologs are sorted according to their similarity score. For an orthology statement additional analysis are required.
The data basis for the orthologs is OrthoDB v9 containing OGS3 for Tribolium castaneum and 2015_04 (r6.07) for Drosophila melanogaster.

Closest fly homologs

The homolog genes are determined by a protein blast against Drosophila with a cutoff of E < 10-5 and no limitation of the required coverage. The three most significant hits are listed on the details page.

Detailed gene information

The link(s) opens a new window with detailed information about the gene(s) targeted by the iB-Nr. The detailed information includes the cDNA, mRNA and peptid sequence.

Tron details page

Number of links of the selected node and all descending ones

The printed number is the amount of iBeetle data sets, which are linked to the current node and the downstream ones. For the downstream search the class hierarchy as well as the part_of relation ship is used. The later one is not displayed in the tree on the left, so that not all involved nodes can be seen directly in the ontology tree. For the caluclation of the number the same nodes are taken into account as in the algorithm for the "Search for morphological defects" (without developmental stage or any other selections made)
For example the descending nodes for adult_leg are:

  • adult_coxa
  • adult_cuticle_of_mesothoracic_leg
  • adult_femur
  • adult_leg
  • adult_mesothoracic_coxa
  • adult_mesothoracic_femur
  • adult_mesothoracic_tarsomere
  • adult_mesothoracic_tarsus
  • adult_metathoracic_femur
  • adult_metathoracic_leg
  • adult_metathoracic_pretarsus
  • adult_metathoracic_tarsus
  • adult_metathoracic_tibia
  • adult_pretarsus
  • adult_prothoracic_leg
  • adult_prothoracic_tarsomere
  • adult_prothoracic_tarsus
  • adult_tarsomere
  • adult_tarsus
  • adult_tibia

The list of hyperlinks belong all to the selected node.