A gene-based high-resolution comparative radiation hybrid map as a framework for genome sequence assembly of a bovine chromosome 6 region associated with QTL for growth, body composition, and milk performance traits

BACKGROUND: A number of different quantitative trait loci (QTL) for various phenotypic traits, including milk production, functional, and conformation traits in dairy cattle as well as growth and body composition traits in meat cattle, have been mapped consistently in the middle region of bovine chromosome 6 (BTA6). Dense genetic and physical maps and, ultimately, a fully annotated genome sequence as well as their mutual connections are required to efficiently identify genes and gene variants responsible for genetic variation of phenotypic traits. A comprehensive high-resolution gene-rich map linking densely spaced bovine markers and genes to the annotated human genome sequence is required as a framework to facilitate this approach for the region on BTA6 carrying the QTL. RESULTS: Therefore, we constructed a high-resolution radiation hybrid (RH) map for the QTL containing chromosomal region of BTA6. This new RH map with a total of 234 loci including 115 genes and ESTs displays a substantial increase in loci density compared to existing physical BTA6 maps. Screening the available bovine genome sequence resources, a total of 73 loci could be assigned to sequence contigs, which were already identified as specific for BTA6. For 43 loci, corresponding sequence contigs, which were not yet placed on the bovine genome assembly, were identified. In addition, the improved potential of this high-resolution RH map for BTA6 with respect to comparative mapping was demonstrated. Mapping a large number of genes on BTA6 and cross-referencing them with map locations in corresponding syntenic multi-species chromosome segments (human, mouse, rat, dog, chicken) achieved a refined accurate alignment of conserved segments and evolutionary breakpoints across the species included. CONCLUSION: The gene-anchored high-resolution RH map (1 locus/300 kb) for the targeted region of BTA6 presented here will provide a valuable platform to guide high-quality assembling and annotation of the currently existing bovine genome sequence draft to establish the final architecture of BTA6. Hence, a sequence-based map will provide a key resource to facilitate prospective continued efforts for the selection and validation of relevant positional and functional candidates underlying QTL for milk production and growth-related traits mapped on BTA6 and on similar chromosomal regions from evolutionary closely related species like sheep and goat. Furthermore, the high-resolution sequence-referenced BTA6 map will enable precise identification of multi-species conserved chromosome segments and evolutionary breakpoints in mammalian phylogenetic studies

Development and Validation of Clinical Whole-Exome and Whole-Genome Sequencing for Detection of Germline Variants in Inherited Disease

Context.-With the decrease in the cost of sequencing, the clinical testing paradigm has shifted from single gene to gene panel and now whole-exome and whole-genome sequencing. Clinical laboratories are rapidly implementing next-generation sequencing-based whole-exome and whole-genome sequencing. Because a large number of targets are covered by whole-exome and whole-genome sequencing, it is critical that a laboratory perform appropriate validation studies, develop a quality assurance and quality control program, and participate in proficiency testing. Objective.-To provide recommendations for wholeexome and whole-genome sequencing assay design, validation, and implementation for the detection of germline variants associated in inherited disorders. Data Sources.-An example of trio sequencing, filtration and annotation of variants, and phenotypic consideration to arrive at clinical diagnosis is discussed. Conclusions.-It is critical that clinical laboratories planning to implement whole-exome and whole-genome sequencing design and validate the assay to specifications and ensure adequate performance prior to implementation. Test design specifications, including variant filtering and annotation, phenotypic consideration, guidance on consenting options, and reporting of incidental findings, are provided. These are important steps a laboratory must take to validate and implement whole-exome and whole-genome sequencing in a clinical setting for germline variants in inherited disorders

Computational Modeling of the Human Microbiome

The impact of microorganisms on human health has long been acknowledged and studied, but recent advances in research methodologies have enabled a new systems-level perspective on the collections of microorganisms associated with humans, the human microbiome. Large-scale collaborative efforts such as the NIH Human Microbiome Project have sought to kick-start research on the human microbiome by providing foundational information on microbial composition based upon specific sites across the human body. Here, we focus on the four main anatomical sites of the human microbiome: gut, oral, skin, and vaginal, and provide information on site-specific background, experimental data, and computational modeling. Each of the site-specific microbiomes has unique organisms and phenomena associated with them; there are also high-level commonalities. By providing an overview of different human microbiome sites, we hope to provide a perspective where detailed, site-specific research is needed to understand causal phenomena that impact human health, but there is equally a need for more generalized methodology improvements that would benefit all human microbiome research

Haplotype estimation in polyploids using DNA sequence data

Polyploid organisms possess more than two copies of their core genome and therefore contain k>2 haplotypes for each set of ordered genomic variants. Polyploidy occurs often within the plant kingdom, among others in important corps such as potato (k=4) and wheat (k=6). Current sequencing technologies enable us to read the DNA and detect genomic variants, but cannot distinguish between the copies of the genome, each inherited from one of the parents. To detect inheritance patterns in populations, it is necessary to know the haplotypes, as alleles that are in linkage over the same chromosome tend to be inherited together. In this work, we develop mathematical optimisation algorithms to indirectly estimate haplotypes by looking into overlaps between the sequence reads of an individual, as well as into the expected inheritance of the alleles in a population. These algorithm deal with sequencing errors and random variations in the counts of reads observed from each haplotype. These methods are therefore of high importance for studying the genetics of polyploid crops. </p

Genomic comparisons and genome architecture of divergent Trypanosoma species

Virulent Trypanosoma cruzi, and the non-pathogenic Trypanosoma conorhini and Trypanosoma rangeli are protozoan parasites with divergent lifestyles. T. cruzi and T. rangeli are endemic to Latin America, whereas T. conorhini is tropicopolitan. Reduviid bug vectors spread these parasites to mammalian hosts, within which T. rangeli and T. conorhini replicate extracellularly, while T. cruzi has intracellular stages. Firstly, this work compares the genomes of these parasites to understand their differing phenotypes. Secondly, genome architecture of T. cruzi is examined to address the effect of a complex hybridization history, polycistronic transcription, and genome plasticity on this organism, and study its highly repetitive nature and cryptic genome organization. Whole genome sequencing, assembly and comparison, as well as chromosome-scale genome mapping were employed. This study presents the first comprehensive whole-genome maps of Trypanosoma, and the first T. conorhini strain ever sequenced. Original contributions vii to knowledge include the ~21-25 Mbp assembled genomes of the less virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E, containing ~10,000 to 13,000 genes, and the ~36 Mbp genome assembly of highly virulent T. cruzi CL with ~24,000 genes. The T. cruzi strains exhibited ~74% identity to proteins of T. rangeli or T. conorhini. T. rangeli and T. conorhini displayed greater complex carbohydrate metabolic capabilities, and contained fewer retrotransposons and multigene family copies, e.g. mucins, DGF-1, and MASP, compared to T. cruzi. Although all four genomes appear highly syntenic, T. rangeli and T. conorhini exhibited greater karyotype conservation. T. cruzi genome architecture studies revealed 66 maps varying from 0.13 to 2.4 Mbp. At least 2.6% of the genome comprises highly repetitive repeat regions, and 7.4% exhibits repetitive regions barren of labels. The 66 putative chromosomes identified are likely diploid. However, 20 of these maps contained regions of up to 1.25 Mbp of homology to at least one other map, suggestive of widespread segmental duplication or an ancient hybridization event that resulted in a genome with significant redundancy. Assembled genomes of these parasites closely reflect their phylogenetic relationships and give a greater context for understanding their divergent lifestyles. Genome mapping provides insight on the genomic evolution of these parasites

Rotation invariant visual processing for spatial memory in insects

Visual memory is crucial to navigation in many animals, including insects. Here, we focus on the problem of visual homing, that is, using comparison of the view at a current location with a view stored at the home location to control movement towards home by a novel shortcut. Insects show several visual specializations that appear advantageous for this task, including almost panoramic field of view and ultraviolet light sensitivity, which enhances the salience of the skyline. We discuss several proposals for subsequent processing of the image to obtain the required motion information, focusing on how each might deal with the problem of yaw rotation of the current view relative to the home view. Possible solutions include tagging of views with information from the celestial compass system, using multiple views pointing towards home, or rotation invariant encoding of the view. We illustrate briefly how a well-known shape description method from computer vision, Zernike moments, could provide a compact and rotation invariant representation of sky shapes to enhance visual homing. We discuss the biological plausibility of this solution, and also a fourth strategy, based on observed behaviour of insects, that involves transfer of information from visual memory matching to the compass system

Quantitative methods for profiling dynamic chromatin features

Living systems, from entire organisms down to the single cells constituting them are dynamic entities that continuously adapt and respond to their local environment. Cells achieve this through gene expression programs derived from static information encoded in the DNA made dynamic through chemical modifications at the chromatin level, collectively termed the epigenome. Numerous epigenetic regulators have been implicated in early embryonic developmental transitions and pluripotency. Ex vivo, the different states of pluripotency can be recapitulated by embryonic stem cells (ESCs) grown in defined media conditions. Many developmental gene promoters in ESCs display co-occurrence of the activating histone H3 lysine 4 trimethylation (H3K4me3) mark and the repressive H3K27me3 mark. This distinctive bivalent signature is considered to poise expression, allowing timely resolution to an active or inactive state depending on the signal. The distribution of histone modifications and chromatin-associated factors across the genome can be mapped using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). However, traditional ChIP-seq methods fail to quantitatively profile the nuanced global and local epigenetic rewiring that takes place in key developmental stages. This thesis addresses this limitation through the development of a quantitative multiplexed ChIP-seq technology: MINUTE (multiplexed indexed unique molecule T7 amplification end to end sequencing) ChIP. Across the three papers included in this thesis, we reveal the underpinnings of chromatin state dynamics in early mouse and human embryonic development by employing MINUTE ChIP. In Paper I, we first show that MINUTE ChIP enables accurate quantitative comparisons over a wide linear range. By employing it to characterize mouse ESCs grown in 2i and serum conditions, we find that the 2i naïve state is characterized by high global levels of H3K27me3 and low H3K4me3. At bivalent promoters, we observe that while H3K27me3 levels are stably maintained between serum and 2i, H3K4me3 levels are higher in the serum condition. Through quantitative epigenome profiling, in Paper II we find that naïve human ESCs also have broad global gain of Polycomb repressive complex 2 (PRC2)-mediated H3K27me3 and define a previously unrecognized, naïve-specific set of bivalent promoters. Bulk and single-cell transcriptomics confirmed that naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state which is resolved to an active state upon depletion of H3K27me3. Therefore, we discovered that PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development. In paper III we show how quantitative RNA polymerase II occupancy profiles generated by MINUTE ChIP can be integrated with transient transcriptomics data to unravel genome wide transcriptional kinetics in three mESCs pluripotent states: naïve, ground and paused. Taken together, this thesis provides compelling evidence for a broad H3K27me3 hypermethylation of the genome in both naïve mouse and human ESCs and the basis for substantially revising the model for bivalency during embryonic developmen

Metabolomics Data Processing and Data Analysis—Current Best Practices

Metabolomics data analysis strategies are central to transforming raw metabolomics data files into meaningful biochemical interpretations that answer biological questions or generate novel hypotheses. This book contains a variety of papers from a Special Issue around the theme “Best Practices in Metabolomics Data Analysis”. Reviews and strategies for the whole metabolomics pipeline are included, whereas key areas such as metabolite annotation and identification, compound and spectral databases and repositories, and statistical analysis are highlighted in various papers. Altogether, this book contains valuable information for researchers just starting in their metabolomics career as well as those that are more experienced and look for additional knowledge and best practice to complement key parts of their metabolomics workflows

Development of an experimental and computational platform for enhanced characterization of modified peptides and proteins in environmental proteomics

Over the last decade, mass spectrometry based proteomics has been established as the front-runner in systems-level protein expression studies. However, with the field progressing into research of more and more complex samples, novel challenges have been raised with respect to efficient protein extraction and computational matching. In this dissertation, various aspects in the proteomics workflow, including experimental and computational approaches, have been developed, optimized and systematically evaluated. In this work, some of the critical factors with respect to proteomics sample preparation, like available biomass, detergent removal methods, and intact protein fractionation to achieve deeper proteome measurements were evaluated. The presented work will help the broader scientific community to carefully design proteomics experiments especially in biomass limited samples. A second major area of focus in this dissertation is comprehensive characterization of post-translational modifications (PTMs) in different biological systems. PTMs are critical for functioning of both the prokaryotic and eukaryotic species and this dissertation will highlight some of the experimental strategies to explore the diversity of PTMs in microbial isolates via application of alternate protease and multiple fragmentation schemes. The PTM discovery approach will be further extended into a complex eukaryotic model trees species Populus trichocarpa using recently developed sequence tagging methods to carryout broad scale PTM search and a complete blind PTM search. Although the work presented in this dissertation mainly revolves around prokaryotic and eukaryotic species involved in environmental proteomics, the general considerations outlined in this work can be extended to every proteomics pipeline. Thus this dissertation will benefit the scientific community in carefully designing experiments before embarking on any research project involving mass spectrometry