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.