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.
","abstract_html":"<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>","abstract_has_math":false,"creators":["Moore, Brad T."],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Baugh, Ryan"],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017","date_published":"2017","updated_at":"2026-07-24T02:06:56Z","subjects":["Genetics","Bioinformatics","Molecular biology","caenorhabditis elegans","mos1","population sequencing","quantitative genetics","transposon sequencing"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/10161/14559","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Baugh, Ryan"]},{"key":"dc:creator","label":"Author","values":["Moore, Brad T."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2017-05-16T17:29:02Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2019-04-24T08:17:07Z"]},{"key":"dc:date.issued","label":"Date","values":["2017"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Genetics","Bioinformatics","Molecular biology","caenorhabditis elegans","mos1","population sequencing","quantitative genetics","transposon sequencing"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10161/14559"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Population Sequencing for Studying Natural and Artifcial Variation in C. elegans"]}]}],"canonical_facts":{"dc:contributor.advisor":["Baugh, Ryan"],"dc:creator":["Moore, Brad T."],"dc:date.accessioned":["2017-05-16T17:29:02Z"],"dc:date.available":["2019-04-24T08:17:07Z"],"dc:date.issued":["2017"],"dc:description.abstract":["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"],"dc:subject":["Genetics","Bioinformatics","Molecular biology","caenorhabditis elegans","mos1","population sequencing","quantitative genetics","transposon sequencing"],"dc:title":["Population Sequencing for Studying Natural and Artifcial Variation in C. elegans"],"dc:type":["Dissertation"]},"updated_at":"2026-07-24T02:06:56Z"}