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Clone mapper: an online suite of tools for RNAi experiments in Caenorhabditis elegans.

Thakur N, Pujol N, Tichit L, Ewbank JJ - G3 (Bethesda) (2014)

Bottom Line: 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 show that Clone Mapper overcomes the limitations of existing techniques and provide examples illustrating its potential for the identification of biologically relevant genes.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université, Case 906, 13288 Marseille Cedex 9, France INSERM U1104, 13288 Marseille, France CNRS UMR7280, 13288 Marseille, France.

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An example of RNAi clone identification using Clone Mapper. The DNA sequences obtained upon sequencing of 10 RNAi clones, from (Zugasti et al. 2014), were used as input into Clone Mapper. The results obtained, ranked by “Aligned region,” are shown in this screen-grab. The leftmost column shows the library name of each clone, the next column the name of the clone that best matches the experimentally determined RNAi clone insert sequence. In this example, half the clones appeared to be what was expected; for 3 of 5 of the others, an alternative identity was assigned with high confidence. For the remaining clones only a very short sequence matches a clone in the in silico library. These sequences can be compared directly to the genome of C. elegans by clicking the link in the rightmost column. The exact meaning of the different columns and options is explained in the help document, accessible by clicking the question mark at the top of the screen.
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fig3: An example of RNAi clone identification using Clone Mapper. The DNA sequences obtained upon sequencing of 10 RNAi clones, from (Zugasti et al. 2014), were used as input into Clone Mapper. The results obtained, ranked by “Aligned region,” are shown in this screen-grab. The leftmost column shows the library name of each clone, the next column the name of the clone that best matches the experimentally determined RNAi clone insert sequence. In this example, half the clones appeared to be what was expected; for 3 of 5 of the others, an alternative identity was assigned with high confidence. For the remaining clones only a very short sequence matches a clone in the in silico library. These sequences can be compared directly to the genome of C. elegans by clicking the link in the rightmost column. The exact meaning of the different columns and options is explained in the help document, accessible by clicking the question mark at the top of the screen.

Mentions: Given the errors that are intrinsic to any large collection of clones, it is indispensable to verify that RNAi clones selected through screens correspond to what they are supposed to be. This is generally done by resequencing and comparing the obtained sequence to the genome of C. elegans and crosschecking the position with that expected for the clone. Checking in this way becomes laborious when one needs to sequence-verify tens or hundreds of clones. We therefore made a BLASTN-based tool to match experimentally determined clone sequences with our in silico clone sequence libraries. It returns an output showing whether the clone is the expected one, and if not what the clone is most likely to be (Figure 3). This became the first tool in a suite that we have called Clone Mapper and for which we provide a web-based access via www.ciml.univ-mrs.fr/EWBANK_jonathan/software.html. The other functionalities are described below.


Clone mapper: an online suite of tools for RNAi experiments in Caenorhabditis elegans.

Thakur N, Pujol N, Tichit L, Ewbank JJ - G3 (Bethesda) (2014)

An example of RNAi clone identification using Clone Mapper. The DNA sequences obtained upon sequencing of 10 RNAi clones, from (Zugasti et al. 2014), were used as input into Clone Mapper. The results obtained, ranked by “Aligned region,” are shown in this screen-grab. The leftmost column shows the library name of each clone, the next column the name of the clone that best matches the experimentally determined RNAi clone insert sequence. In this example, half the clones appeared to be what was expected; for 3 of 5 of the others, an alternative identity was assigned with high confidence. For the remaining clones only a very short sequence matches a clone in the in silico library. These sequences can be compared directly to the genome of C. elegans by clicking the link in the rightmost column. The exact meaning of the different columns and options is explained in the help document, accessible by clicking the question mark at the top of the screen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232539&req=5

fig3: An example of RNAi clone identification using Clone Mapper. The DNA sequences obtained upon sequencing of 10 RNAi clones, from (Zugasti et al. 2014), were used as input into Clone Mapper. The results obtained, ranked by “Aligned region,” are shown in this screen-grab. The leftmost column shows the library name of each clone, the next column the name of the clone that best matches the experimentally determined RNAi clone insert sequence. In this example, half the clones appeared to be what was expected; for 3 of 5 of the others, an alternative identity was assigned with high confidence. For the remaining clones only a very short sequence matches a clone in the in silico library. These sequences can be compared directly to the genome of C. elegans by clicking the link in the rightmost column. The exact meaning of the different columns and options is explained in the help document, accessible by clicking the question mark at the top of the screen.
Mentions: Given the errors that are intrinsic to any large collection of clones, it is indispensable to verify that RNAi clones selected through screens correspond to what they are supposed to be. This is generally done by resequencing and comparing the obtained sequence to the genome of C. elegans and crosschecking the position with that expected for the clone. Checking in this way becomes laborious when one needs to sequence-verify tens or hundreds of clones. We therefore made a BLASTN-based tool to match experimentally determined clone sequences with our in silico clone sequence libraries. It returns an output showing whether the clone is the expected one, and if not what the clone is most likely to be (Figure 3). This became the first tool in a suite that we have called Clone Mapper and for which we provide a web-based access via www.ciml.univ-mrs.fr/EWBANK_jonathan/software.html. The other functionalities are described below.

Bottom Line: 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 show that Clone Mapper overcomes the limitations of existing techniques and provide examples illustrating its potential for the identification of biologically relevant genes.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université, Case 906, 13288 Marseille Cedex 9, France INSERM U1104, 13288 Marseille, France CNRS UMR7280, 13288 Marseille, France.

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