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

dc.contributor.advisor

Baugh, L Ryan

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Moore, Brad T.

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2017-05-16T17:29:02Z

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2019-04-24T08:17:07Z

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2017

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Computational Biology and Bioinformatics

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The advent of high coverage and low cost sequencing technologies has allowed for

newer and more powerful approaches in molecular and population genetics. Transposon

sequencing, where genome-saturated mutant populations allele frequencies are

measured before and after selection, functionally characterizes each and every gene

in the genome in a single experiment. The approach has been successfully applied

to a variety of phenotypes in a variety of unicellular systems: growth and motility

in E. coli, synthetic genetic interactions in yeast, and in vitro pathogen-resistance in

mammalian cell lines. However, transposon insertion typically produces null alleles,

which can be valuable to identify gene function, but evolutionary insight relies on

identifcation of naturally occurring polymorphisms affecting the trait of interest.

Genome-wide association studies (GWAS) can be used to study the effect of natural

genetic variation on a trait, but they grow prohibitively expensive if the number of

individuals to genotype and phenotype becomes large.

Here I describe the application of transposon sequencing and pooled sequencing

GWAS in the whole metazoan model, Caenorhabditis elegans. Transposon sequencing

has not been previously implemented in an animal model. I have sequenced a control

library using our method, C. elegans transposon sequencing (CeTnSeq). We have

constructed a new Mos1 transposon mutator strain that is more convenient to use

than the existing strain and allows for extra-chromosomal insertions to be degraded

by restriction digest. My preliminary results show that our method is qualitatively

effective at identifying transposon insertion sites, but suffers from PCR duplication

error. I propose to optimize the number of PCR cycles in the library and to include

unique molecular identifiers (UMI) in the library adaptor. I also show that the

restriction digest is effective at removing extra-chromosomal array insertions from

the library.

I constructed simulation models to help design optimal Ce-TnSeq experiments

with respect to statistical power for a proposed starvation survival assay. I considered

many parameters affecting the design, including: culture size, number of generations,

expected effect size, sequencing coverage, and sample size. I show that the number

of homozygous mutant animals in the screen is a critical factor in the design of

experiments. I also saw diminishing returns with respect to increasing sample size

and sequencing depth. These simulations will be invaluable in designing future Ce-

TnSeq experiments and identifying critical aspects of the protocol to optimize.

We performed pooled sequencing (using restriction-site associated DNA sequencing)

on a population of 95 wild isolates subjected to starvation. I identified strains

that were resistant and sensitive to starvation, and we verified these results using

traditional methods. We used our population sequencing data to perform an association

study of starvation survival across the 95 strains, and identified two statistically

significant quantitative trait loci.

dc.identifier.uri

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

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Genetics

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Bioinformatics

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Molecular biology

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Caenorhabditis elegans

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Genetics

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mos1

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Population sequencing

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Quantitative genetics

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transposon sequencing

dc.title

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

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Dissertation

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23

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