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- Creators: Kusumi, Kenro
- Creators: School of Mathematical and Natural Sciences
Description
Well-established model systems exist in four out of the seven major classes of vertebrates. These include the mouse, chicken, frog and zebrafish. Noticeably missing from this list is a reptilian model organism for comparative studies between the vertebrates and for studies of biological processes unique to reptiles. To help fill in this gap the green anole lizard, Anolis carolinensis, is being adapted as a model organism. Despite the recent release of the complete genomic sequence of the A. carolinensis, the lizard lacks some resources to aid researchers in their studies. Particularly, the lack of transcriptomic resources for lizard has made it difficult to identify genes complete with alternative splice forms and untranslated regions (UTRs). As part of this work the genome annotation for A. carolinensis was improved through next generation sequencing and assembly of the transcriptomes from 14 different adult and embryonic tissues. This revised annotation of the lizard will improve comparative studies between vertebrates, as well as studies within A. carolinensis itself, by providing more accurate gene models, which provide the bases for molecular studies. To demonstrate the utility of the improved annotations and reptilian model organism, the developmental process of somitogenesis in the lizard was analyzed and compared with other vertebrates. This study identified several key features both divergent and convergent between the vertebrates, which was not previously known before analysis of a reptilian model organism. The improved genome annotations have also allowed for molecular studies of tail regeneration in the lizard. With the annotation of 3' UTR sequences and next generation sequencing, it is now possible to do expressional studies of miRNA and predict their mRNA target transcripts at genomic scale. Through next generation small RNA sequencing and subsequent analysis, several differentially expressed miRNAs were identified in the regenerating tail, suggesting miRNA may play a key role in regulating this process in lizards. Through miRNA target prediction several key biological pathways were identified as potentially under the regulation of miRNAs during tail regeneration. In total, this work has both helped advance A. carolinensis as model system and displayed the utility of a reptilian model system.
ContributorsEckalbar, Walter L (Author) / Kusumi, Kenro (Thesis advisor) / Huentelman, Matthew (Committee member) / Rawls, Jeffery (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2012
Description
Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. We describe the establishment of primary dermal fibroblasts cells lines from 28 autopsy donors. These fibroblasts were used to examine the proliferative effects of establishment protocol, tissue amount, biopsy site, and donor age. As proof-of-principle, iPSCs were generated from fibroblasts from a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. To our knowledge, this is the first study describing autopsy donor-derived somatic cells being used for iPSC generation and subsequent neural differentiation. This unique approach also enables us to compare iPSC-derived cell cultures to endogenous tissues from the same donor. We utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, supported by (i) a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain, (ii) an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue, and (iii) a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. These studies support the utility of autopsy donors' somatic cells for iPSC-based neurological disease models, and provide evidence that in vitro neural differentiation can result in physiologically progression.
ContributorsHjelm, Brooke E (Author) / Craig, David W. (Thesis advisor) / Wilson-Rawls, Norma J. (Thesis advisor) / Huentelman, Matthew J. (Committee member) / Mason, Hugh S. (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2013
Description
With increasing urbanization, organisms face a myriad of novel ecological challenges. While the eco-evolutionary dynamics of urbanization are currently receiving a great deal of attention, the effect of urban disturbance on the microbiome of urban organisms is relatively unstudied. Indeed, studies of the microbiome may illuminate the mechanisms by which some species thrive after urbanization (pest implications), while other species go locally extinct (biodiversity implications). We investigated the gut microbiome of the Western black widow spider (Latrodectus hesperus). L. hesperus is an ideal model system as they are a pest species of medical importance in urban ecosystems, often forming dense urban infestations relative to the sparse populations found in their native Sonoran Desert. To gain insight into the composition of the microbiome in L. hesperus and its potential function, we sampled 4 urban, 4 desert, and 2 laboratory-reared spiders, and high-throughput sequencing of the 16S rRNA V4 region was used to investigate the diversity of gut microbiota. Dominant bacterial phyla across all samples were Firmicutes, Proteobacteria, and Actinobacteria. While desert widows showed more gut microbial diversity than urban widows, the difference was not statistically significant. The relative abundance of taxonomic classes Blastocatellia, Acidobacteriia, and Thermoleophilia detected in desert spiders was especially higher than those in urban and laboratory-reared spiders. However, urban spiders had a higher relative abundance of taxonomic class Actinomycetia. Differences in widow gut microbiome diversity improves our understanding of how features unique to a habitat, like prey diversity and soil microbes, may be shaping their microbiome. Additionally, this work further highlights the impact urbanization has on biodiversity loss, which indirectly develops a new biomarker for differentiating between urban and desert black widow spiders based on their gut microbiome.
ContributorsAsrari, Hasti (Author) / Johnson, Chad (Thesis director) / Sandrin, Todd (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor) / School of Life Sciences (Contributor)
Created2022-12
Description
The HLA, Human Leukocyte Antigens, are encoded by a polymorphic set of genes where even a single base change can impact the function of the body’s immune response to foreign antigens [1]. Although many methods exist to type these alleles using whole-genome sequencing (WGS), few can use RNA sequencing (RNA-seq) to show the functional expression of the alleles with its inconsistency in coverage, and none of these allow for novel allele discovery. We present an approach using partially ordered graphs to project sequenced data onto the known alleles allowing for accurate and efficient typing of the HLA genes with flexibility for discovering new alleles and tolerance for poor sequence quality. This graph-guided approach to assembling and typing the HLA genes from RNA-seq has applications throughout precision medicine, facilitating the prevention and treatment of autoimmune diseases where allele expression can change. It is also a necessary step for determining donors for organ transplants with the least likelihood of rejection. This novel approach of combining database matching with partially ordered graphs for assembling genetic sequences of RNA-seq data could be applied towards typing other alleles.
ContributorsMallett, Shayna (Author) / Lee, Heewook (Thesis director) / Wilson, Melissa (Committee member) / Barrett, The Honors College (Contributor) / Computer Science and Engineering Program (Contributor)
Created2022-05
Description
The use of DNA testing has been focused primarily on biological samples such as blood or saliva found at crime scenes. These types of evidence in the forensic field are sometimes difficult to come by, especially when there is no body to find to verify things such as identity or status of a person. In the case of the burial of a body, they can be remote and relocated multiple times depending on each situation. Clandestine burials are not uncommon especially in the Arizona desert by the United States and Mexico border. Since there is no physical body to find the next best avenue to finding a clandestine burial is through search teams which can take weeks to months or other expensive technology such as ground penetrating radar (GPR). A new more interesting avenue to search for bodies is using the most found material–soil. Technology has allowed the possibility of using soil DNA microbiome testing initially to study the varieties of microbes that compose in soil. Microbiomes are unique and plentiful and essentially inescapable as humans are hosts of millions of them. The idea of a microbiome footprint at a crime scene seems out of reach considering the millions of species that can be found in various areas. Yet it is not impossible to get a list of varieties of species that could indicate there was a body in the soil as microbiomes seep through from decomposition. This study determines the viability of using soil microbial DNA as a method of locating clandestine graves by testing 6 different locations of a previous pig decomposition simulation. These two locations give two different scenarios that a body may be found either exposed to the sun in an open field or hidden under foliage such as a tree in the Sonoran Desert. The experiment will also determine more factors that could contribute to a correlation of microbiome specific groups associated with decomposition in soil such as firmicutes. The use of soil microbial DNA testing could open the doors to more interpretation of information to eventually be on par with the forensic use of biological DNA testing which could potentially supplement testimonies on assumed burial locations that occurs frequently in criminal cases of body relocation and reburial.
ContributorsMata Salinas, Jennifer (Author) / Marshall, Pamela (Thesis director) / Bolhofner , Katelyn (Committee member) / Wang, Yue (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Natural Sciences (Contributor) / School of Humanities, Arts, and Cultural Studies (Contributor)
Created2022-05
Description
Damage to the central nervous system due to spinal cord or traumatic brain injury, as well as degenerative musculoskeletal disorders such as arthritis, drastically impact the quality of life. Regeneration of complex structures is quite limited in mammals, though other vertebrates possess this ability. Lizards are the most closely related organism to humans that can regenerate de novo skeletal muscle, hyaline cartilage, spinal cord, vasculature, and skin. Progress in studying the cellular and molecular mechanisms of lizard regeneration has previously been limited by a lack of genomic resources. Building on the release of the genome of the green anole, Anolis carolinensis, we developed a second generation, robust RNA-Seq-based genome annotation, and performed the first transcriptomic analysis of tail regeneration in this species. In order to investigate gene expression in regenerating tissue, we performed whole transcriptome and microRNA transcriptome analysis of regenerating tail tip and base and associated tissues, identifying key genetic targets in the regenerative process. These studies have identified components of a genetic program for regeneration in the lizard that includes both developmental and adult repair mechanisms shared with mammals, indicating value in the translation of these findings to future regenerative therapies.
ContributorsHutchins, Elizabeth (Author) / Kusumi, Kenro (Thesis advisor) / Rawls, Jeffrey A. (Committee member) / Denardo, Dale F. (Committee member) / Huentelman, Matthew J. (Committee member) / Arizona State University (Publisher)
Created2015
Description
Agassiz’s desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.
Data Availability: All genomic and transcriptomic sequence files are available from the NIH-NCBI BioProject database (accession numbers PRJNA352725, PRJNA352726, and PRJNA281763). All genome assembly, transcriptome assembly, predicted protein, transcript, genome annotation, repeatmasker, phylogenetic trees, .vcf and GO enrichment files are available on Harvard Dataverse (doi:10.7910/DVN/EH2S9K).
ContributorsTollis, Marc (Author) / DeNardo, Dale F (Author) / Cornelius, John A (Author) / Dolby, Greer A (Author) / Edwards, Taylor (Author) / Henen, Brian T. (Author) / Karl, Alice E. (Author) / Murphy, Robert W. (Author) / Kusumi, Kenro (Author)
Created2017-05-31