A C. elegans Fosmid Library A C. elegans Fosmid Library

Jaryn Perkins1,2, Kim Wong3, René Warren3, Jacquie Schein3, Jeff Stott3, Rob Holt3, Steve Jones3, Marco Marra3, and Don Moerman1,2

1Michael Smith Laboratories, U.B.C., Vancouver, B.C., Canada V6T 1Z4
2Department of Zoology, University of British Columbia, B.C. Vancouver,Canada V6T 1Z4
3Genome Sciences Centre, BC Cancer Agency, Vancouver, B.C. Canada, V5Z 4S6

The availability of genomic clones with 35 to 40kb inserts has been a critical resource to the C. elegans community. Fingerprinted and ordered cosmid clones harboring such inserts were the backbone of the sequencing project. Further utility was found when individual research labs were trying to map mutants. Complementing a mutant by injecting a strain with overlapping cosmid clones eventually led to mutation identity. This approach is still widely used and is an important tool for mutational analysis. Many of the cosmid libraries are now 20 years old and there are reports of some of them losing viability. This persuaded us to consider building a new DNA library with total coverage of the genome. We also felt it was best to make a 40kb clone insert library rather than rely on YAC or BAC clone libraries. The large insert clones are not trivial to manipulate and do not in many cases give you the resolution that is required for many experiments.

With these considerations in mind we have chosen to make a fosmid rather than a cosmid library. The cosmid libraries have been excellent workhorses but many clones within the libraries have been prone to rearrangements. By using a fosmid vector (pCC1FOS from Epicentre) that is maintained at low copy number until induced we hope that the occurrence of such rearrangements will be reduced. The use of fosmids as backbones allow for the maintenance of large pieces of DNA (around 40kB) in limited number (1-5) per bacterial host. Up to this point, such a resource was unavailable for C. elegans.

We have created an N2 fosmid library and are currently mapping clones to the genome. We are mapping all fosmid clones to the C. elegans genome by pair-wise alignment of fosmid end-reads. Initial analysis of the library quality shows that the average insert size is ~ 43,284 bp and 86.7% of the clones have paired end-reads with correct relative orientation. Currently, with over 13,307 logical clones sequenced we have obtained approximately 5.74X clone coverage of the genome. Further analysis will allow us to determine gene coverage for the worm. This resource was created for public domain use and is available to interested researchers.

This abstract was presented at the Fifteenth International C. elegans meeting.