Identifying Variants in Neuroligin Pathway Genes Using Next Generation Sequencing Technologies Open Access
Steinberg, Karyn Meltz (2009)
Published
Abstract
The fields of population genomics and evolutionary quantitative
genetics provide a
framework in which one can best pursue the heritable component of
complex human
phenotypes. A central challenge lies in the comprehensive
ascertainment of all the
relevant genomic variation, irrespective of their population
frequency, in a large
collection of human samples. Autism Spectrum Disorder (ASD) is a
complex human
neurodevelopmental disorder, characterized by a high heritability
and a nearly 4:1 male
excess. Applying this comprehensive genomic variation detection
paradigm poses two
main challenges. The first lies in developing and applying
technologies that can
efficiently detect the relevant genomic variation. The second lies
in applying these
methods in the context of a testable genetic hypothesis that might
elucidate the etiology
of ASD. Here I report on a series of studies that have pursued both
of these challenges.
While sequencing technologies have advanced rapidly in the past
decade, the ability to
rapidly isolated target DNA for sequencing has lagged. I first
describe a novel technique
for isolating target DNA for downstream resequencing applications.
This protocol, named
Microarray-based Genomic Selection, is able to efficiently select
user-defined sequence
that is then hybridized to resequencing arrays. Two experiments
that used Microarray-
based Genomic Selection for resequencing are described. In the
first experiment the
technology was used to isolate all of the exons on the X
chromosome, while the second
experiment used it to isolate specific genes in the neuroligin
pathway that are
hypothesized to contribute to ASD. Advantages and limitations of
MGS are discussed. To
address the genetic basis of ASD, I first selected X-linked
neuroligin pathway genes
thought to harbor ASD susceptibility alleles that may help explain
the male excess in
ASD. Using samples of male individuals with ASD obtained from the
Autism Genetic
Resource Exchange (AGRE), I performed paired-end multiplexed
sequencing on the
Illumina Genome Analyzer to comprehensively sequence the genomic
regions containing
the neuroligin pathway genes. This study identified a series of
candidate variants that
may contribute to ASD susceptibility. Finally, I will highlight the
importance of using
quantitative evolutionary genetics when analyzing and interpreting
sequence data.
Table of Contents
DISTRIBUTION AGREEMENT
APPROVAL SHEET
ABSTRACT COVER PAGE
ABSTRACT
COVER PAGE
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
TABLES
FIGURES
I. Introduction
I.I. Quantitative genetics and complex disease
I.I.1. Evolutionary quantitative genetics
I.I.2. Common Disease Common Variant hypothesis
I.I.3. Common Disease Rare Variant hypothesis
I.II. Autism as a complex trait
I.II.1. Patterns of inheritance
I.II.2. The X chromosome and Cognitive Disorders
I.III. Development of sequencing technologies
I.III.1. First generation technology
I.III.2. Second generation technology
I.III.3. Third generation technology
I.IV. Scope of thesis
I.V. Figure Legends
I.VI. Figures
1. Chapter 1
1.1. Abstract
1.2. Text
1.3. Figure Legends
1.4. Supplementary Methods
1.5. Tables
1.6. Figures
2. Chapter 2
2.1. Introduction
2.2. Results
2.2.1. Statistical analysis of MGS probes
2.2.2. Chip redesign
2.3. Discussion
2.4. Methods
2.5. Figure Legends
2.6. Figures
3.1. Introduction
3.1.1. Association of autism with neuroligin genes
3.1.2. Evolutionary history of neuroligins
3.1.3. Mouse models
3.1.4. Further evidence of Xp22.3 involvement in cognitive
disorders
3.2. Results
3.3. Discussion
3.4. Methods
3.4.1. Sample selection
3.4.2. Array design
3.4.3. Target DNA selection and resequencing
3.4.4. Analysis
3.5. Tables
3.6. Figure Legends
3.7. Figures
4. Chapter 4
4.1. Introduction
4.2. Results
4.2.1. Evaluation of Alignment and Assembly Algorithms
4.2.2. Annotation of Variants
4.2.3. Indel Analysis
4.3. Discussion
4.4. Conclusion
4.5. Methods
4.5.1. Sample Selection
4.5.2. Primer Design
4.5.3. Long PCR
4.5.4. Fragmentation
4.5.5. End Repair
4.5.6. Add "A" Bases to 3' End of Fragments
4.5.7. Ligation of Adapters
4.5.8. Size Selection and Enrichment
4.5.9. Cluster Generation and Paired End Multiplexed
Sequencing
4.5.10. Data Analysis
4.6. Tables
4.7. Figure Legends
4.8. Figures
5. Conclusion
6. References
About this Dissertation
School | |
---|---|
Department | |
Degree | |
Submission | |
Language |
|
Research Field | |
Keyword | |
Committee Chair / Thesis Advisor | |
Committee Members |
Primary PDF
Thumbnail | Title | Date Uploaded | Actions |
---|---|---|---|
|
Identifying Variants in Neuroligin Pathway Genes Using Next Generation Sequencing Technologies () | 2018-08-28 11:20:50 -0400 |
|
Supplemental Files
Thumbnail | Title | Date Uploaded | Actions |
---|