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Journal Articles G3 Year : 2014

Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans


RNA interference (RNAi), mediated by the introduction of a specific double-stranded RNA, is a powerful method to investigate gene function. It is widely used in the Caenorhabditis elegans research community. An expanding number of laboratories conduct genome-wide RNAi screens, using standard libraries of bacterial clones each designed to produce a specific double-stranded RNA. Proper interpretation of results from RNAi experiments requires a series of analytical steps, from the verification of the identity of bacterial clones, to the identification of the clones' potential targets. Despite the popularity of the technique, no user-friendly set of tools allowing these steps to be carried out accurately, automatically, and at a large scale, is currently available. We report here the design and production of Clone Mapper, an online suite of tools specifically adapted to the analysis pipeline typical for RNAi experiments with C. elegans. We show that Clone Mapper overcomes the limitations of existing techniques and provide examples illustrating its potential for the identification of biologically relevant genes. The Clone Mapper tools are freely available via KEYWORDS database algorithm gene discovery functional genomics MPscan RNA interference (RNAi) is a powerful and widely used method to investigate gene function. Researchers using the model nematode Caenorhabditis elegans often use a feeding method for RNAi that involves culturing worms on a bacterial clone expressing a double-stranded RNA (dsRNA) that is intended to target a specific worm gene (Timmons et al. 2001; Timmons and Fire 1998). Because worms can be handled robotically, screens can be automated and large numbers of clones tested in parallel (Squiban et al. 2012). Collections of RNAi clones are available. One made by the Ahringer lab contains polymerase chain reaction (PCR)-amplified fragments of genomic DNA (Kamath et al. 2003), whereas the library made by the Vidal lab (Rual et al. 2004) was constructed from ORFeome clones, which are derived from cDNA (Reboul et al. 2001). Part of the strength of the method arises from the fact that knowledge of the sequence of the dsRNA in principle allows the corresponding target gene(s) to be identified. In common with any large-scale resource, the available bacterial RNAi clone libraries contain errors (e.g., clone positions inverted on 96-well plates). For the Ahringer library, this error rate is estimated to be approximately 7% ( pages/rnai.html; Qu et al. 2011). These can be compounded by handling errors during a screen, resulting in error rates as high as 15% (Pukkila-Worley et al. 2014). This means that clones need to be checked by sequencing to confirm their identity. Interpreting the sequences, to confirm clone identity, can be laborious when dealing with large numbers of clones. In C. elegans long dsRNAs (often .1 kb) are used, in contrast to the short interfering RNAs (siRNA; typically 19225 bp long) used in vertebrates. Each dsRNA can thus give rise to a multitude of siRNAs, which complicates target identification. Many published studies have relied on the assignment of targets provided by the community database Wormbase (Yook et al. 2012). This currently suffers from a number of limitations (Wormbase release WS242). The first is that target identification is based on empirical criteria. The sequence of a " primary target " is at least 95% identical with the clone insert sequence for at least 100 nucleotides (Fievet et al. 2013); for " secondary targets " the
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hal-01243281 , version 1 (14-12-2015)



Nishant Thakur, Nathalie Pujol, Laurent Tichit, Jonathan J. Ewbank. Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans. G3, 2014, 4, pp.2137-2145. ⟨10.1534/g3.114.013052⟩. ⟨hal-01243281⟩
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