Population Sequencing for Studying Natural and Artifcial Variation in C. elegans

<p>The advent of high coverage and low cost sequencing technologies has allowed for</p><p>newer and more powerful approaches in molecular and population genetics. Transposon</p><p>sequencing, where genome-saturated mutant populations allele frequencies are</p><p>measured before and after selection, functionally characterizes each and every gene</p><p>in the genome in a single experiment. The approach has been successfully applied</p><p>to a variety of phenotypes in a variety of unicellular systems: growth and motility</p><p>in E. coli, synthetic genetic interactions in yeast, and in vitro pathogen-resistance in</p><p>mammalian cell lines. However, transposon insertion typically produces null alleles,</p><p>which can be valuable to identify gene function, but evolutionary insight relies on</p><p>identifcation of naturally occurring polymorphisms affecting the trait of interest.</p><p>Genome-wide association studies (GWAS) can be used to study the effect of natural</p><p>genetic variation on a trait, but they grow prohibitively expensive if the number of</p><p>individuals to genotype and phenotype becomes large.</p><p>Here I describe the application of transposon sequencing and pooled sequencing</p><p>GWAS in the whole metazoan model, Caenorhabditis elegans. Transposon sequencing</p><p>has not been previously implemented in an animal model. I have sequenced a control</p><p>library using our method, C. elegans transposon sequencing (CeTnSeq). We have</p><p>constructed a new Mos1 transposon mutator strain that is more convenient to use</p><p>than the existing strain and allows for extra-chromosomal insertions to be degraded</p><p>by restriction digest. My preliminary results show that our method is qualitatively</p><p>effective at identifying transposon insertion sites, but suffers from PCR duplication</p><p>error. I propose to optimize the number of PCR cycles in the library and to include</p><p>unique molecular identifiers (UMI) in the library adaptor. I also show that the</p><p>restriction digest is effective at removing extra-chromosomal array insertions from</p><p>the library.</p><p>I constructed simulation models to help design optimal Ce-TnSeq experiments</p><p>with respect to statistical power for a proposed starvation survival assay. I considered</p><p>many parameters affecting the design, including: culture size, number of generations,</p><p>expected effect size, sequencing coverage, and sample size. I show that the number</p><p>of homozygous mutant animals in the screen is a critical factor in the design of</p><p>experiments. I also saw diminishing returns with respect to increasing sample size</p><p>and sequencing depth. These simulations will be invaluable in designing future Ce-</p><p>TnSeq experiments and identifying critical aspects of the protocol to optimize.</p><p>We performed pooled sequencing (using restriction-site associated DNA sequencing)</p><p>on a population of 95 wild isolates subjected to starvation. I identified strains</p><p>that were resistant and sensitive to starvation, and we verified these results using</p><p>traditional methods. We used our population sequencing data to perform an association</p><p>study of starvation survival across the 95 strains, and identified two statistically</p><p>significant quantitative trait loci.</p>

Author dc:creator

• Moore, Brad T.

Advisor dc:contributor.advisor

• Baugh, Ryan

• Genetics
• Bioinformatics
• Molecular biology
• caenorhabditis elegans
• mos1
• population sequencing
• quantitative genetics
• transposon sequencing

Handle dc:identifier.uri

https://hdl.handle.net/10161/14559

OAI identifier oai:identifier

oai:dukespace.lib.duke.edu:10161/14559