Tyson Fuller & Nikale Pettie to Present at Genetics Student Seminar 12/5/17

Tyson’s Abstract:

Learning the Epilepsy Alphabet from A (syne1a&b) to Z (zfhx3)

Epilepsy, which affects ~1% of the population, is caused by abnormal synchronous neural activity in the central nervous system (CNS). While there is a significant genetic contribution to epilepsy, the underlying causes for the majority of genetic cases remain unknown. The NIH Undiagnosed Diseases Project (UDP) utilized exome sequencing to identify genetic variants in patients affected by various conditions with undefined etiology, including epilepsy. Confirming the functional relevance of the candidate genes identified by exome sequencing in a timely manner is crucial to translating exome data into clinically useful information. To this end, I developed a high throughput version of a seizure-sensitivity assay in zebrafish (Danio rerio) to rapidly evaluate candidate genes found by exome sequencing. This assay uses pentylenetetrazol (PTZ) to induce seizures in zebrafish larvae and the motility tracking software of the Zebrabox (Viewpoint Life Sciences) is utilized to record each larva’s total movement (cm). This generates massive data sets. Therefore, I developed an open access software, SEIZR (Studying Epilepsy In Zebrafish using R), to rapidly and efficiently analyze the data.  My project focuses on characterizing the functional role of fifteen genes in the NIH UDP for which mutations have been associated with epilepsy, and for which zebrafish orthologues have been identified. Using SEIZR, I characterized all fifteen candidate genes in the context of seizure sensitization. Here, I show the findings of two genes, syne1b and zfhx3, both of which result in seizure sensitization when knocked down in the zebrafish. I show that each of these genes is expressed in regions of the brain involved in motor control during critical times of neural development. Further, I find syne1b knockdown results in axon defects in the retina. Reagents to test for rescue and localization are in progress.

Nikale’s Abstract:

Filling the Gaps in the Drosophila Yakuba Genome

Most ‘reference’ genomes are incomplete, with sequence gaps throughout the euchromatic genomic regions of model organisms such as Drosophila melanogaster, zebrafish, mice and even humans. These sequence gaps are the result of difficulties in cloning and/or sequence assembling across simple repeats and repetitive elements. Because these gap regions can contain genes and add uncertainty to physical and genetic distances, the presence of gaps can hinder mapping strategies (QTL, GWAS, etc.) as well as evolutionary studies.  The genome of our model organism of interest, D. yakuba, is completely sequenced but it also contains genomic gaps. Most short-read sequencing technologies (i.e., Illumina) are usually unable to address the gaps but the more recent long-read Pacific Biosciences (PacBio) SMRT sequencing approach provides a solution to this problem because individual reads can be 5-20kb long and hence span the entirety of challenging genomic regions. On the negative side, PacBio reads have an error rate that is much higher than (classic) Sanger and Illumina sequencing. The ideal strategy is, therefore, a ‘hybrid’ sequencing approach where PacBio sequences are used to fill in the gaps to later take advantage of deep-coverage Illumina sequencing to ensure high quality sequences for these new genomic regions. We used this hybrid method to improve the D. yakuba genome. We fill more than 60% of the gaps and we add approximately 6.2 million bp of high quality sequence to the D. yakuba reference genome, creating an improved version of the original reference genome with an additional 6% of genomic data. The new D. yakuba genome will be useful in several downstream applications such as identifying novel genes and transcripts.  In particular, studies that use genomic distance, such as the calculation of recombination frequency per nucleotide as well as the prediction of the impact of selection on diversity will be much more accurate.