1. Where can I obtain my clone of interest from?
All ORFs marked as "cloned" by the ORFeome project can be obtained through "Horizon Discovery" (c-elegans-orf-collection, c-elegans-rnai-collection, c-elegans-promoter-collection).

2. The C. elegans ORFeome represents pools of ORFs, what does that mean?
After PCR amplification with gene-specific primers, a pool of wildtype as well as mutated PCR products (of all splice-variants of that ORF present in the cDNA library) are cloned into the Gateway vector. In order to obtain a wild-type sequence of each splice-variant of your gene of interest, single cells have to be picked and their vectors sequenced.

3. Why have some predicted ORFs been cloned in both ORFeome version 1 and 3? For most part, only ORFs that could not be cloned in ORFeome version 1 or that were cloned out of frame in ORFeome version 1, and that furthermore showed an updated exon/intron structure at the 5' and/or 3'end between version WS9 and WS100 of Wormbase were part of the ORFeome version 3 dataset. We also attempted to clone a small number of ORFs that were already cloned in frame in ORFeome version 1 but for which new predictions exist.

4. What is the definition of ORFeome version 1, 1.1, 2, 3 and 3.1 ?
ORFeome version 1:
attempt to clone all WS9 ORFs
ORFeome version 1.1:
ORFs cloned in ORFeome version 1
ORFeome version 1.1a:
ORFs cloned in-frame in ORFeome version 1
ORFeome version 1.1b:
ORFs cloned out-of-frame in ORFeome version1
ORFeome version 2:
attempt to isolate a resequenced wild-type sequence for each splice-variant of each v1.1a ORF (Reboul et al, 2003)
ORFeome version 3:
attempt to clone all repredicted and new WS100 ORFs
ORFeome version 3.1:
the complete collection of ORFs cloned in-frame in ORFeome version 1 and 3

5. What kind of data has been used to update ORF predictions between Wormbase versions 9 and 100? Wormbase curators mainly used EST, OST, cDNA and RNA data to curate the C. elegans genome. Other factors such as comparative genomics, SL sequences, Blast results or the overlap of a predicted ORF with a newly annotated non-coding region or a modified neighboring gene helped reannotate the C. elegans genome. Comparative genomics certainly represents the most promising genome annotation tool for C. elegans in the near future. Although only one additional nematode, C. briggsae, has been sequenced so far, the sequences of 3 additional nematode species is underway and will represent a powerful predictor of C. elegans ORF structures.

6. Is there a size limit of ORFs that can be cloned into the Gateway vector? The largest ORF that we were able to cloned into the Gateway vector is 6kb.

7. What is the advantage of cloning ORFs using the Gateway recombinational cloning system? The main advantage of using a recombinational cloning system is the ease with which each ORF can be transferred from the Entry Vector into any vector of choice.

8. Do those ORFs that have not been cloned actually exist, are they wrong predictions? We believe that most ORFs that we were not able to clone so far exist but have been mispredicted at their 3’ or 5’ ends. In fact most gene prediction tools have difficulties predicting gene boundaries as opposed to internal exons. Using internal primers we were able to clone more than 80% of a subset of ORFs that could not be cloned in ORFeome version 1 and 56% of a subset of ORFs that could not be cloned in ORFeome version 3. This indicates that even after 2 rounds of cloning and 4 years of constant efforts to improve the genome annotation, many uncloned C. elegans ORFs exist but couldn’t be cloned due to mispredictions at their boundaries.

9. Does Wormbase take into account structural differences found between OSTs and predictions? All the structural differences observed between OSTs and gene predictions in ORFeome version 1 have been analyzed by the curators of Wormbase. OSTs that showed a different gene structure but no frame problem have either replaced the old prediction or been added as a splice variant. Predicted ORFs whose corresponding OSTs showed a frame problem have have been curated.

10.How come the OST corresponding to my cloned ORF is only partially overlapping that ORF? The OST displayed in the alignment image of each clone-information page represents the available sequence obtained by sequencing the clone at both ends. In case of long clones the Forward and Reverse tags don't overlap and provide an only partial confirmation of the predicted gene structure. This is also true when only one of the two sequence tags is of good enough quality to be aligned to it's ORF. By clicking on the "See OST" link on each clone-information page, we show the complete sequence of each OST that wereceived for that clone (even those OSTs that did not pass the quality treshold to be aligned by Acembly).