Next Generation Sequencing Guided SNP Mapping

Abstract

Oogenesis is dependent on precise translational control and localization of numerous morphogens within the oocyte to achieve faithful patterning. Gurken, (Grk) is one such protein and is responsible for specification of the dorsal/ ventral axis. Mutations in the spindle-B gene results in inefficient gurken translation due to activation of a meiotic DNA damage checkpoint. This checkpoint activation inhibits the Vasa RNA helicase, an essential grk translation factor. Without proper Gurken levels, the egg chambers develop defects, the most severe being complete ventralization. A 2004 forward genetic screen targeting the 3rd chromosome identified thirty nine unique mutants in a spn-BBU mutant background. Two of these lines had already been mapped, the other lines were screened for their ability to suppress the ventralized spn-BBU phenotype and therefore stimulate grk translation. Eggs laid by homozygotes from each of the isogenized lines were scored for their dorsal/ventral polarity and compared to those of the control group of spn-BBU homozygotes. We have taken advantage of a next-generation sequencing approach to identify candidate mutations in 10 independent lines from a forward genetic screen for regulators of dorsal ventral patterning during Drosophila oogenesis. Through a partnership with Roswell Park Cancer Institute in Buffalo, NY, the best suppressor lines were subject to whole-genome re-sequencing using the Illumina HiSeq 2000 platform. A large scale mapping experiment was started, creating recombinant flies for six of the lines. After multiple universal markers were developed to distinguish these chromosomes from the mapping line, a focus was placed on one of the suppressor lines, CA231. A previous mapping experiment on this line placed the mutation toward the end of the right arm. A higher density map was made for this area. The screen was limited by the number of recombinants that showed variation in this area. While the causative mutation has yet to be found, the pool of candidate mutations has been vastly diminished. Furthermore, additional focused mapping projects have been started from the recombinants made in this experiment, using a subset of the markers that are shared with CA231 as a starting point.