Evaluation of a New Method for Large-Scale and Gene-targeted Next Generation DNA Sequencing in Nonmodel Species

The efficient method called exon capture provides for sequencing genes genome-wide, targeting candidate genes, and sampling specific exons within genes. Although developed for model species with available whole genome sequences, the method can capture exons in nonmodel species using the genomic resources of a related model species. How close the relatives must be for effective exon capture is not known. The work herein demonstrates cross-taxa capture in ungulates, using the domestic cow genome as a reference. It also describes a computer program designed for collecting exon sequences for exon capture, allowing users to set per-gene and overall base pair (bp) limits, and to prefer internal or external exons. Cross-taxa exon capture was tested with subject-reference divergence times from 0 to ~60 million years. Sequencing success decreased with increasing subject-reference phylogenetic divergence. With the domestic cow genome as reference, American bison exons, at 1-2 million years (MY) of divergence, were captured as successfully as those of a domestic cow. The cow and bison captures each yielded sequence from ~80% of the 3.6 million bp targeted. Two bighorn sheep, 7 mule deer, and 4 pigs at about 20, 30, and 60 MY of divergence from the cow, respectively, yielded averages of ~70%, ~60%, and ~55% of the targeted bp. A gene family with many closely related, duplicated loci was expected to show reduced success compared to the whole collection. This prediction was supported, as 63 exons in the MHC gene family sequences yielded 62% fully sequenced in the cow, and 32%, 20%, and 4% for the bighorn, deer, and pigs, respectively. A comparison of two sequence alignment programs showed that Stampy, designed for high sample-reference divergence, was dramatically better than BWA, designed for low divergence, only in the pig capture, in which Stampy yielded ~30% more bp than did BWA. A universal ungulate exon capture array could be developed using the 8,999 exons that were fully sequenced in all species, including the pig at ~60 MY. As this method helps us understand the genetic basis of evolutionary processes, so it can contribute to an informed study and stewardship of our ecological endowment

Crop improvement studies based on molecular approaches in interspecific Oil palm hybrids

162 p.Oil Palm (OP) is the crop with the highest oil yield per hectare and as a result, its use has spread rapidly in tropical regions of Asia, Africa and America. The main OP plantations consist of Elaeis guineensis (Eg) species, known to produce high amounts of oil. However, in American regions this species is being affected by the ¿Pudrición de Cogollo¿ disease leading to dead palms. Therefore, OP companies started crossing this species with E. oleifera (Eo) palms which is resistant to this disease. The obtained interspecific hybrids show interesting characteristic inherited from both parents; resistance to different diseases, interesting oil quality characteristics, competitive oil production and decreased height which prolongs its useful life. However, little work has been done in the improvement of these hybrids. This thesis tries to address this gap applying different molecular approaches. First, an extensive study of an amplicon of the ¿Shell-thickness¿ (Sh) gene has been conducted on 568 Eg, Eo and hybrid genotypes. Then, with the aim to discover promising new Candidate Genes (CG) that could be exploited in further molecular assisted selection systems (MAS) a large phenotypic study of 25 production and quality traits have been performed within 198 hybrid genotypes fllowed by two Association Mapping (AM) assays. These latter have been based on targeted CG and random Restriction site associated RNA sequencing(RARSeq) approaches.Neiker teknalia Lafabril Energy & Palma Sampoerna Agr

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises.Defense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006)United States. National Institutes of Health (NIH AI100190

Identifying novel genes associated with response to nicotine in a zebrafish model of drug dependence.

PhDTobacco addiction is a leading preventable cause of death worldwide and places a heavy social and financial burden on society. There exists a substantial genetic variability in smoking behavior, the mechanisms of which are largely unknown. Despite significant advances in sequencing power, progress in the identification of genetic variants affecting smoking behavior based on human genome wide association studies has been slow. Thus this thesis investigates the utility of zebrafish as a model species in which to search for genetic variants affecting nicotine seeking. The work is based on the premise that as zebrafish are vertebrate with conserved neurochemical pathways and circuitry with humans, and the pathways involved in drug mediated reward and addiction are evolutionarily ancient, homologues of genes affecting zebrafish nicotine-seeking behavior will likely affect human smoking behavior. Thus results in zebrafish can be used to direct human genetic studies. The first result chapter addresses the hypothesis that zebrafish show conserved reward responses to common drugs of abuse. A conditioned place preference assay is used to assess zebrafish reward responses to stimulants, opioids, benzodiazepines and alcohol. The results indicate that, with the exception of benzodiazepines, reward responses are conserved, supporting the use of this model in a screen for genetic variants affecting nicotine preference. The second and third results chapters describe the findings of a pilot screen of ENU-mutagenized zebrafish provided by the Sanger Institute, Cambridge. I demonstrate that nicotine preference is heritable in fish as in Abstract 5 humans and identify 3 mutant lines that show increased or decreased nicotine place preference. Genotyping indicated that one of the families showing increased nicotine preference carries a predicted loss of function mutation in the slit3 gene. The involvement of this gene in nicotine preference was confirmed in a separate line. Further characterization of this line using qPCR showed slit3 mutants to have altered developmental expression of key nicotinic and dopaminergic genes. Having identified the slit3 gene as a locus affecting nicotine seeking in fish, I then tested the hypothesis that results in fish could be used to predict loci that affect human smoking behavior. Cohorts of patients were genotyped for 20 SNPs within the slit3 locus. Results of this analysis identified 1 novel SNP in the slit3 gene associated with smoking behavior in a cohort of individuals that were heavy smokers. This result was validated in cohorts of low and normal smoking prevalence. These data demonstrate the utility of behavioral assays in zebrafish to identify genes affecting human behavior and pave the way for the use of zebrafish to inform human studies exploring the genetic basis of drug seeking and behavioral disease.Medical Research Council, grant number G1000403

Time to Diagnosis and Persistence: The Two Major Determinants of Effective Tuberculosis Control

The greatest challenge confronting effective tuberculosis (TB) eradication is the time to diagnosis, and duration of treatment of chronically infected individuals which represent a pool of infection. In an attempt to help limit the spread of TB in New Zealand, a fast SNP based diagnostic test was developed, to quickly identify the highly transmissible and virulent endemic Rangipo strain. The role of VapBC toxin-antitoxin systems in M. tuberculosis has been the subject of great interest recently, due to their expanded number in the genome and links with virulence and the regulation of cell growth in response to environmental stress. Their ability to regulate growth under adverse conditions for presumed survival advantages possibly leading to dormancy or persistence, make them ideal candidates for the development of new M. tuberculosis treatments. To establish differential expression of vapC, and therefore identify possible pathways and functions of the VapBC proteins, RT-qPCR was used to assess the expression levels of vapB and vapC in M. smegmatis under conditions of stress. No consistant changes in vapC mRNA levels were observed, resulting in the hypothesis that it is not the transcriptional differences which are important in the regulation of VapC, but post-transcriptional factors. In order to investigate the function(s) of M. tuberculosis VapBCs, these VapBC proteins were expressed and purified in M. smegmatis, and the VapC toxin tested for RNase activity. The purification, expression, RNase testing and bioinformatic analysis of M. tuberculosis VapCs suggested that VapCRv2530c, VapCRv0065 and VapCRv0617 may all target the same recognition sequence, UA*GG. Bioinformatic analysis revealed an abundance of this target sequence in horizontal gene transfer and TA genes, raising the possibility that VapC toxins could be functioning as selfish elements, or initiating transcriptional regulation cascades when a rapid change in the proteomic response and metabolic state of the cell is required. It is intriguing that the three M. tuberculosis VapC proteins tested thus far appear to target the same recognition sequence, possibly suggesting that all 47 VapCs are RNases and are targeting the same sequence. Alternatively; VapCs may belong to sub-groups targeting different sequences, allowing M. tuberculosis to exude both gross and fine metabolic control; or, they may share the same target, but are regulated by different activators triggered in response to different environmental stimuli

Development of new genome-informed genotyping tools for Aphanomyces astaci

Aphanomyces spp. are water moulds, eukaryotic fungus-like organisms, belonging to the class Oomycota. This genus contains primary pathogens of plants and animals as well as opportunistic and saprotrophic species. One of the animal parasites (A. astaci) is the causal agent of the crayfish plague, a disease listed by the World Organisation for Animal Health (OIE). It is believed that A. astaci was first introduced into Italy from the US in the late 19th century and rapidly spread in Europe causing the decline of native crayfish. It currently threatens to wipe out the UK native white-clawed crayfish (Austropotamobius pallipes). Random amplified polymorphic DNA (RAPD-PCR) on pure isolates of A. astaci distinguished five genotypes (A, B, C, D, and E). This distinction proved to be a useful tool for epidemiological studies aimed at understanding the history and spread of the disease in Europe; furthermore, there are differences in virulence among genotypes. No discriminatory morphological or physiological characters are available and widely used markers such as the internal transcribed spacer (ITS), the divergent domains regions (D1-D2) of nuclear large subunit (LSU) rDNA, and cytochrome c oxidase subunit I (COI) also fail to discriminate between A. astaci genotypes. There are some practical drawbacks to genotype by the currently available genotyping methods. Whole genome sequencing (WGS) was used to catalogue DNA single nucleotide variants and genotype-unique genomic regions that could be exploited as phylogenetic markers. These newly developed molecular markers were tested both on pure cultures and historical samples derived from outbreaks and carrier crayfish available in our laboratories, validating these genotyping methods, which represent new diagnostic tools aiding the detection and prevention of crayfish plague

Clinical Applications of Pharmacogenomics of Warfarin

Ph.DDOCTOR OF PHILOSOPH