Copy number variants and selective sweeps in natural populations of the house mouse (Mus musculus domesticus)

Copy–number variants (CNVs) may play an important role in early adaptations, potentially facilitating rapid divergence of populations. We describe an approach to study this question by investigating CNVs present in natural populations of mice in the early stages of divergence and their involvement in selective sweeps. We have analyzed individuals from two recently diverged natural populations of the house mouse (Mus musculus domesticus) from Germany and France using custom, high–density, comparative genome hybridization arrays (CGH) that covered almost 164 Mb and 2444 genes. One thousand eight hundred and sixty one of those genes we previously identified as differentially expressed between these populations, while the expression of the remaining genes was invariant. In total, we identified 1868 CNVs across all 10 samples, 200 bp to 600 kb in size and affecting 424 genic regions. Roughly two thirds of all CNVs found were deletions. We found no enrichment of CNVs among the differentially expressed genes between the populations compared to the invariant ones, nor any meaningful correlation between CNVs and gene expression changes. Among the CNV genes, we found cellular component gene ontology categories of the synapse overrepresented among all the 2444 genes tested. To investigate potential adaptive significance of the CNV regions, we selected six that showed large differences in frequency of CNVs between the two populations and analyzed variation in at least two microsatellites surrounding the loci in a sample of 46 unrelated animals from the same populations collected in field trappings. We identified two loci with large differences in microsatellite heterozygosity (Sfi1 and Glo1/Dnahc8 regions) and one locus with low variation across the populations (Cmah), thus suggesting that these genomic regions might have recently undergone selective sweeps. Interestingly, the Glo1 CNV has previously been implicated in anxiety–like behavior in mice, suggesting a differential evolution of a behavioral trai

Lifetime Surface Phosphor Thermometry - Technique Developments, Sources of Error, and Applications

Temperature is not only a fundamental aspect of everyday life, but it is also an essential metric for thermal conversion systems concerning efficiency, emissions, and component lifetime estimation. Therefore, a vast range of different methods to measure temperature has been developed. The technique of focus in this thesis is lifetime-based surface phosphor thermometry.In phosphor thermometry, one leverages the changes in luminescence of materials called phosphors to measure temperature. The spectral changes can result from shifts in the relative intensity of emission lines in the spectra or shifts in emission line wavelength with temperature. In addition, changes in the lifetime of the phosphorescence signal with temperature can be utilized for thermometry. Inorganic phosphors are ceramic substances that can operate with a range of luminescence activators. Most often, the luminescence originates from trivalent lanthanide ions or transition metal ions doped in a host crystal.Surface phosphor thermometry has the benefit of being a remote sensing technique, where one coats a phosphor on a surface of interest. This phosphor coating is then excited with a remote source, for example, pulsed laser light, and the phosphor luminescence is detected remotely. In this thesis, technology developments, error sources analysis, and applications of phosphor thermometry are presented. The technology developments present a high-temperature calibration system and a method for analyzing the lifetime components in the luminescence decay of phosphors in detail. An analysis of upconversion phosphors for temperature measurements beneath thermal barrier coatings and an investigation of high-temperature phosphors to 1900 K and their sensitivities to the oxygen content of the gas environment are also included in this work.The investigations into potential error sources include how PMT nonlinearity effects can influence the measured lifetime with excitation frequency from 10 Hz up to 10 kHz. In addition, the phosphor luminescence decay time impact of high repetition excitation was also investigated and analyzed to see potential error sources for temperature measurements.Phosphor thermometry was also used in two applications, including measuring the surface temperature of burning wood pellets and the surface temperature of a combustion nozzle in a down-scaled gas turbine combustor using hydrogen-enriched methane as fuel

Development of Mechanosensory Innervation in the Frog, Xenopus Laevis

ABSTRACT DEVELOPMENT OF MECHANOSENSORY INNERVATION IN THE FROG, XENOPUS LAEVIS by Peter Feuk The University of Wisconsin-Milwaukee, 2017 Under the Supervision of Dr. R. David Heathcote This study aims to investigate whether a specific target cell in the epidermis of the African clawed frog, Xenopus laevis, guides the initial outgrowth and pattern of Rohon-Beard (RB) cells and their survival. RB cells are primary mechanosensory neurons present during the early developmental stages of X. laevis. These neurons provide sensory input to the frog throughout embryonic and larval development before initiating apoptosis around the start of metamorphosis. The innervation of embryonic skin cells by RB neurons exhibits a distinct pattern that features encircling of a specific subset of epidermal cells. We hypothesize that encircled cells could be a recently discovered cell type that synthesizes and secretes serotonin. To test whether these small secretory cells (SSC) play a chemoattractive role in the innervation of primary mechanosensory neurons in X. laevis, we used a variety of approaches. By immunolabeling RB processes in the epidermis, we established the wild-type pattern and quantity of innervation. Gain of function and loss of function tests utilizing pharmacological modulators of serotonin, directly examined the role of SSCs. We showed that serotonin levels can be over or under expressed, and that there is a quantifiable response of the resulting mechanosensory innervation. RB neurons innervating larval skin lacking serotonin showed a lower frequency of encircled SSCs as well as a greater number of retraction bulbs. We also showed a correlation between behavior and serotonin levels in the epidermis. Loss of function treatments showed a higher proportion of animals failing to respond to stimuli and an increase in inappropriate escape responses. Finally, we related changes in X. laevis larval skin and mechanosensory neurons during metamorphosis to tissue remodeling and the transition to adult sensory function. This research provides insight into axonal guidance and the patterning of mechanosensory innervation in X. laevis. It stands as an example of how the innervation of the vertebrate skin is established and maintained between developmental stages

Global and unbiased detection of splice junctions from RNA-seq data

SplitSeek can be used to detect novel splicing events in SOLiD RNA-seq data without the need for a pre-defined library

Efficient TALEN-mediated gene knockout in livestock

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications

A novel class of miniature inverted repeat transposable elements (MITEs) that contain hitchhiking (GTCY)n microsatellites

The movement of miniature inverted repeat transposable elements (MITEs) modifies genome structure and function. We describe the microsatellite-associated interspersed nuclear element 2 (MINE-2), that integrates at consensus WTTTT target sites, creates dinucleotide TT target site duplications (TSDs), and forms predicted MITE-like secondary structures; a 5\u27 subterminal inverted repeat (SIR; AGGGTTCCGTAG) that is partially complementary to a 5\u27 inverted repeat (IR; ACGAAGCCCT) and 3\u27-SIRs (TTACGGAACCCT). A (GTCY)(n) microsatellite is hitchhiking downstream of conserved 5\u27MINE-2 secondary structures, causing flanking sequence similarity amongst mobile microsatellite loci. Transfection of insect cell lines indicates that MITE-like secondary structures are sufficient to mediate genome integration, and provides insight into the transposition mechanism used by MINE-2s

The Diploid Genome Sequence of an Individual Human

Presented here is a genome sequence of an individual human. It was produced from ~32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2-206 bp), 292,102 heterozygous insertion/deletion events (indels)(1-571 bp), 559,473 homozygous indels (1-82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information

ESTIMATING GENOME-WIDE COPY NUMBER USING ALLELE SPECIFIC MIXTURE MODELS

Genomic changes such as copy number alterations are thought to be one of the major underlying causes of human phenotypic variation among normal and disease subjects [23,11,25,26,5,4,7,18]. These include chromosomal regions with so-called copy number alterations: instead of the expected two copies, a section of the chromosome for a particular individual may have zero copies (homozygous deletion), one copy (hemizygous deletions), or more than two copies (amplifications). The canonical example is Down syndrome which is caused by an extra copy of chromosome 21. Identification of such abnormalities in smaller regions has been of great interest, because it is believed to be an underlying cause of cancer. More than one decade ago comparative genomic hybridization (CGH)technology was developed to detect copy number changes in a high-throughput fashion. However, this technology only provides a 10 MB resolution which limits the ability to detect copy number alterations spanning small regions. It is widely believed that a copy number alteration as small as one base can have significant downstream effects, thus microarray manufacturers have developed technologies that provide much higher resolution. Unfortunately, strong probe effects and variation introduced by sample preparation procedures have made single-point copy number estimates too imprecise to be useful. CGH arrays use a two-color hybridization, usually comparing a sample of interest to a reference sample, which to some degree removes the probe effect. However, the resolution is not nearly high enough to provide single-point copy number estimates. Various groups have proposed statistical procedures that pool data from neighboring locations to successfully improve precision. However, these procedure need to average across relatively large regions to work effectively thus greatly reducing the resolution. Recently, regression-type models that account for probe-effect have been proposed and appear to improve accuracy as well as precision. In this paper, we propose a mixture model solution specifically designed for single-point estimation, that provides various advantages over the existing methodology. We use a 314 sample database, constructed with public datasets, to motivate and fit models for the conditional distribution of the observed intensities given allele specific copy numbers. With the estimated models in place we can compute posterior probabilities that provide a useful prediction rule as well as a confidence measure for each call. Software to implement this procedure will be available in the Bioconductor oligo packagehttp://www.bioconductor.org)

On the power and the systematic biases of the detection of chromosomal inversions by paired-end genome sequencing

One of the most used techniques to study structural variation at a genome level is paired-end mapping (PEM). PEM has the advantage of being able to detect balanced events, such as inversions and translocations. However, inversions are still quite difficult to predict reliably, especially from high-throughput sequencing data. We simulated realistic PEM experiments with different combinations of read and library fragment lengths, including sequencing errors and meaningful base-qualities, to quantify and track down the origin of false positives and negatives along sequencing, mapping, and downstream analysis. We show that PEM is very appropriate to detect a wide range of inversions, even with low coverage data. However, % of inversions located between segmental duplications are expected to go undetected by the most common sequencing strategies. In general, longer DNA libraries improve the detectability of inversions far better than increments of the coverage depth or the read length. Finally, we review the performance of three algorithms to detect inversions -SVDetect, GRIAL, and VariationHunter-, identify common pitfalls, and reveal important differences in their breakpoint precisions. These results stress the importance of the sequencing strategy for the detection of structural variants, especially inversions, and offer guidelines for the design of future genome sequencing projects