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Forward Genetics Tools C. briggsae

Contents:
RFLP Assays
PLP Assays (small indels)
Indel Assays
Genotyping Primers
Insertional Mutagenesis
Repeat Library

It is our goal to develop resources to assist the development of C. briggsae as a model organism. The primary resource that we are constructing is a genetic map to complement the genome sequence; the latter was completed in 2003. In the course of genetic mapping, we have generated assays and other resources that may be of interest to the nematode research community.

We screened the latest build of HK104 SNPs for substitutions that altered the recognition sequence of restriction enzymes. To make this a practical resource, we limited the analysis to 30 inexpensive, reliable restriction enzymes from REBASE. Using our standard PCR protocols (products of 500-1000 bp and primer TM's of 54-56), we generated 2,133 RFLP assays. The full set is available on the data downloads page.

The first version of this resource contained 34 PLP assays based on indels detected by CrossMatch and Ssaha-DIP. We have since obtained the latest version of SSAHA and used it to identify HK104-AF16 indels of at least 7 bp. We also adjusted assay design parameters so that PCR products are 200-400 bp. The latest version (v3.0) contains physical coordinates for 424 small indels based on the new "cb3" genome assembly on Wormbase, which is by chromosome . Version 2.0 includes both physical map (based on Wormbase ultracontigs) and genetic map information for each marker. These assays are currently being validated by our collaborators, and are available on the data downloads page. If you use them for mapping, please send us your results!

We developed an algorithm called BreakPointRead that uses BLAST alignments of sequencing reads to detect putative structural variation. Though still undergoing refinement, the latest BreakPointRead algorithm identified 635 structural variants in HK104. Of these, 157 were classified as insertions or deletions of 50-2,000 bp. We developed PCR assays for these and cross-referenced them to existing maps, ending up with 94 assays for indels positioned on both genetic (v3.3) and physical (cb3) maps. These assays are are available on the data downloads page. If you use them for mapping, please send us your results!

We designed genotyping assays for ~20,000 C. briggsae HK104 SNPs. Each assay includes two PCR primers and 1-2 extension primers for use with the FP-TDI genotyping technology. This is the small- to medium-throughput technology licensed by PerkinElmer that was used to generate data for the genetic map. Assay design details and primer sequence downloads are available on the genotyping primers page.

Transposon-mediated insertional mutagenesis offers an alternative for forward genetics screens, especially those with a large target size. Marie-Anne Felix of the Institut Jacques Monod has applied this technique to C. briggsae using Drosophila Mos transposons. To date she has characterized three Mos1 insertions in strains of C. briggsae.

In the course of assay design, we developed a set of C. briggsae-specific repeats using contributions from WormBase, Repbase, and other sources. The compiled library should be compatible with RepeatMasker and other programs, and can be obtained on the data downloads page.