Generation and application of genomic tools as important prerequisites for sugar beet genome analyses.

Genetic and physical maps of a genome are essential tools for structural, functional and applied genomics. Genetic maps allow the detection of quantitative trait loci (QTLs), the characterisation of QTL effects and facilitate marker-assisted selection (MAS). The characterisation of genome structure and analysis of evolution is augmented by physical maps. Whole genome physical maps or ultimately complete genomic sequences, respectively, of a species display frameworks that provide essential information for understanding processes in respect to physiology, morphology, development and genetics. However, comprehensive annotation underpins the values a genome sequence or physical map represents. An important task of genome annotation is the linkage of genetic traits to the genome sequence, which is facilitated by integrated genetic and physical maps. In the context of this study several sugar beet (Beta vulgaris L.) genomic tools were developed and applied for evolutionary studies and linkage analysis. A new technique allowing high-throughput identification and genotyping of genetic markers was developed, utilising representational oligonucleotide microarray analysis (ROMA). We tested the performance of the method in sugar beet as a model for crop plants with little sequence information available. Genomic representations of both parents of a mapping population were hybridised on microarrays containing custom oligonucleotides based on sugar beet bacterial artificial chromosome (BAC) end sequences (BESs) and expressed sequence tags (ESTs). Subsequent analysis identified potential polymorphic oligonucleotides, which were placed on new microarrays used for screening of 184 F2 individuals. Exploiting known co-dominant anchor markers, we obtained 511 new dominant markers distributed over all nine sugar beet linkage groups and calculated genetic maps. Besides the method´s transferability to other species, the obtained genetic markers will be an asset for ordering of sequence contigs in the context of the ongoing sugar beet genome sequencing project. In addition, possible linkage of physical and genetic maps was provided, since genetic markers were based on source sequences, which were also used for construction of a BAC based physical map utilising a hybridisation approach. An example of the hybridisation based approach for physical map construction and its application for synteny studies was demonstrated. Since little is known about synteny between rosids and Caryophyllales so far, we analysed the extent of synteny between the genomic sequences of two BAC clones derived from two different Beta vulgaris haplotypes and rosid genomes. For selection of the two BAC clones we hybridised 30 oligonucleotide probes based on ESTs corresponding to Arabidopsis orthologs on chromosomes 1 and 4 that were presumably co-localised in the reconstructed Arabidopsis pseudo ancestral genome (Blanc et al. 2003) on sugar beet BAC macroarrays comprising two different sugar beet libraries. A total of 27,648 clones were screened per sugar beet library, corresponding to 4.4-fold and 5.5-fold, respectively, sugar beet genome coverage. We obtained four and five positive clones for the probes on average. Two clones, one from each haplotype that were positive with the same five EST probes, were selected and their genomic sequences were determined, annotated and exploited for synteny studies. Furthermore, I constructed and characterised a sugar beet fosmid library from the doubled haploid accession KWS2320 encompassing 115,200 independent clones. The insert size of the fosmid library was determined by pulsed field gel electrophoresis to be 39 kbp on average, thus representing 5.9-fold coverage of the sugar beet genome. Fosmids bear the advantage of narrowly defined size of the clone inserts, thus fosmid end sequences will essentially contribute to the future assembly and ordering of sequence contigs. Since repeats are a major obstacle for successful assembly of plant genome sequences, frequently causing gaps and misassembled contigs, I generated a genomic short-insert library. The short-insert library facilitated repeat identification within the sugar beet genome, which was exemplarily shown for three miniature inverted-repeat transposable element (MITE) families. Altogether this work contributed substantially to a deeper understanding of the genome structure of sugar beet and provided the basis for successful sequencing of the sugar beet genome

Isolation, Characterization, Mapping and Expression analysis of Porcine Tissue kallikreins

Kallikreins are members of a multigene family of serine proteases that are widespread throughout living organisms. They are found in diverse tissue specific patterns and are known to have highly diverse physiological functions such as fertilization, digestion, regulation of blood flow, blood coagulation, inflammatory responses, endothelial cell migration, tissue remodeling, tumor-cell invasion and programmed cell death as well as disease related specialized enzymatic activities. To gain insight about the structure and evolutionary origins of the kallikrein gene region in the pig, we have isolated several BAC (Bacterial Artificial Chromosome) clones containing members of the porcine tissue kallikrein gene family and constructed a BAC clone based Physical map of the porcine greater kallikrein gene region using hybridization based dot-blot analysis and DNA fingerprinting techniques. We have also mapped the kallikrein gene region in the porcine genome using radiation-hybrid mapping to chromosome 6q1.2. We have further isolated several porcine tissue kallikreins using RT-PCR and conserved sequence information from orthologous human kallikrein genes and have carried out quantitative real-time PCR based, expression analysis to evaluate the expression of tissue kallikreins in the porcine endometrium and conceptus during the estrous cycle and early embryonic development as a first step towards understanding the tissue expression of pig kallikrein gene family members and to better understand the role of kallikreins in placental development and embryonic survival in the pig.Department of Animal Scienc

Focus: A Graph Approach for Data-Mining and Domain-Specific Assembly of Next Generation Sequencing Data

Next Generation Sequencing (NGS) has emerged as a key technology leading to revolutionary breakthroughs in numerous biomedical research areas. These technologies produce millions to billions of short DNA reads that represent a small fraction of the original target DNA sequence. These short reads contain little information individually but are produced at a high coverage of the original sequence such that many reads overlap. Overlap relationships allow for the reads to be linearly ordered and merged by computational programs called assemblers into long stretches of contiguous sequence called contigs that can be used for research applications. Although the assembly of the reads produced by NGS remains a difficult task, it is the process of extracting useful knowledge from these relatively short sequences that has become one of the most exciting and challenging problems in Bioinformatics. The assembly of short reads is an aggregative process where critical information is lost as reads are merged into contigs. In addition, the assembly process is treated as a black box, with generic assembler tools that do not adapt to input data set characteristics. Finally, as NGS data throughput continues to increase, there is an increasing need for smart parallel assembler implementations. In this dissertation, a new assembly approach called Focus is proposed. Unlike previous assemblers, Focus relies on a novel hybrid graph constructed from multiple graphs at different levels of granularity to represent the assembly problem, facilitating information capture and dynamic adjustment to input data set characteristics. This work is composed of four specific aims: 1) The implementation of a robust assembly and analysis tool built on the hybrid graph platform 2) The development and application of graph mining to extract biologically relevant features in NGS data sets 3) The integration of domain specific knowledge to improve the assembly and analysis process. 4) The construction of smart parallel computing approaches, including the application of energy-aware computing for NGS assembly and knowledge integration to improve algorithm performance. In conclusion, this dissertation presents a complete parallel assembler called Focus that is capable of extracting biologically relevant features directly from its hybrid assembly graph

Analysis of the Human T Cell Receptor α/δ Locus: New Approaches to Mapping and Sequencing

The human T cell receptor (TCR) α/δ locus has been mapped and sequenced. This region occupies roughly one megabase (Mb) of DNA or equivalent to one three thousandth of the entire human genome, the longest continuous piece of human DNA yet sequenced. The sequence has provided new insights into the complex organization, structure and evolution of two intermingled multigene families (α and δ), and will hopefully in the future help answer interesting questions concerning the complex expression patterns of TCR α and δ chains and about possible associations between specific polymorphisms in the TCR α/δ locus and susceptibility to autoimmune diseases. Comparison to cDNA data has provided information about expression of each of the TCR elements and about the striking diversification in the third hypervariable or junctional region. The sequence has contributed a glimpse of closely associated genomic DNA, in that the sequences surrounding the TCR locus, include the defender against death gene as well as five olfactory receptor genes. The sequence also harbors many other stretches of DNA, highly similar to previously identified genes, although in most cases, these have been found to be nonfunctional due to one or a few mutations. Comparison of 130 kilobases (kb) in the 3' region of the human sequence with its murine counterpart, suggests this region is highly conserved. The same 3' region has also been found to be limited in the concentration of genome wide repeats compared to the remainder of the locus. Furthermore, it contains a substantially reduced frequency of DNA variations compared to the rest of the locus. Apart from DNA variations in noncoding sequence, polymorphisms have also been identified in the coding regions of the TCR variable (V) gene segments, where, if they lead to amino acid changes, may alter the function of the TCR. During the physical clone mapping and sequencing, new strategies were tested using primarily bacterial artificial chromosome (BAC) clones. These clones proved to be much more reliable and stable than clones currently employed in the human genome project (e.g., cosmids and yeast artificial chromosomes, YACs). BAC inserts can be sequenced completely by the high redundancy shotgun approach. Their insert size, stability, and capacity to be easily sequenced suggests that BAC clones are excellent mapping and sequencing reagents. The ends of BAC clone inserts can be sequenced directly. This has led to the proposal of a new strategy for obtaining the entire DNA sequence of the human genome without physical mapping.</p

Construction of a duck whole genome radiation hybrid panel : an aid for NGS whole genome assembly and a contribution to avian comparative maps

Le canard est une espèce d'importance agronomique en France, principalement à travers l'industrie de foie gras, qui représente plus de 75% de la production mondiale. De plus, c'est aussi un modèle important pour l'étude de l'infection par le virus influenza, pour lequel les oiseaux aquatiques sont un réservoir naturel, car porteurs asymptomatiques. Les travaux réalisés lors de la thèse se situent dans le contexte international de l'étude du génome du canard, comportant la séquence du génome, le séquençage d'EST et l'identification et la cartographie de SNP. Le but à terme pour l'INRA étant de disposer des connaissances sur le génome nécessaires pour la cartographie fine de QTL et l'identification de gènes impliqués dans l'expression de caractères agronomiques. Un panel de 90 d'hybrides irradiés (panel RH) a été réalisé par fusion de cellules donneuses de canard irradiées avec des cellules receveuses de hamster. Afin d'éviter la culture à grande échelle des clones cellulaires, des méthodes de génotypage par PCR utilisant l'amplification complète du génome (WGA) et/ou la réduction des volumes réactionnels ont été testées et deux premières cartes de chromosomes ont ainsi été réalisées. Nous avons également utilisé le génotypage par PCR pour vérifier la qualité de l'assemblage des scaffolds du génome du canard, réalisés par séquençage nouvelle génération Illumina au Beijing Genome Institute (BGI, Chine). Finalement, afin de couvrir le génome complet, nous avons entrepris un séquençage léger (0,1X de profondeur) d'hybrides, permettant une réalisation de cartes plus rapides que par PCR. Ces cartes permettent la détection des réarrangements chromosomiques existant entre les génomes de la poule et du canard, qui sont distants de 80 millions d'années.Duck is a very important agronomic species in France, especially for fatty liver industry which presents 75% worldwide production. Moreover, duck is also a scientific model for avian influenza research as it is a natural reservoir for avian influenza viruses. The work presented here is part of the international collaboration on duck genome sequencing, including SNP detection and mapping, EST sequencing. Our goal is to provide a genome map allowing for fine mapping QTL and identifying candidate genes involved in expression of agronomic traits. A panel composed of 90 radiation hybrids was produced by fusing irradiated duck donor cells with hamster cells. To avoid large-scale culture of the clones, PCR genotyping involving Whole Genome Amplification (WGA) and/or reduction of reaction volumes were tested and two first maps for duck chromosomes were made. We also used the PCR genotyping method to test for the quality of duck sequence scaffold assemblies, which had been produced by the Beijing Genome Institute (BGI, China). Finally, to cover the whole genome, we performed a low-pass sequencing (0.1X depth) of hybrids, allowing for rapid map development. These maps allow the detection of chromosomal rearrangements that have taken place between the duck and chicken genomes, which have diverged 80 million years ago

Whole genome amplification for PGD and PND; molecular and a-CGH diagnosis

Whole genome amplification amplifies the entire genome in a few hours from samples of minimal DNA quantities, even from single cells. This may have many applications, especially in prenatal diagnosis, PGD and PGS. The hypothesis for chapter 3 was: Can multiple displacement amplification (MDA) be used as a universal step prior to molecular analysis for PGD? WGA using MDA (Qiagen) was used on single cells in order to overcome the problem of limited DNA in PGD. MDA allows the diagnosis through haplotyping or a combination of direct and indirect mutation analysis. Different cell types, including buccal cells, lymphocytes, fibroblasts and blastomeres were examined. A modification on the cell lysis buffer was also tested in order to achieve more accurate results. PGD seems to benefit from MDA when multiple tests are performed for direct and indirect analysis. The modified lysis buffer (exclusion of DTT) produced better results than the other lysis buffers and buccal cells do not produce as accurate results as other cell types. The hypothesis was met as the amount of DNA produced by MDA can be used for direct and indirect testing and haplotyping. The hypothesis for chapter 4 was: Is it possible to accurately assess the chromosomes of a single cell by a-CGH? WGA was achieved by MDA and GenomePlex (Sigma) on single lymphocytes, fibroblasts and blastomeres prior to a-CGH analysis. The difficulty of this technique was the high background noise that was produced by WGA that makes interpretation difficult. Different lysis buffers, modifications of the WGA reaction and analysis software were examined for better results. A-CGH slides from different companies and institutions were used. The results showed that GenomePlex produced less background noise compared to MDA but the amplification efficiency of the technique was less reliable. The BlueGnome Cytochip arrays produced the best compared to arrays from any other companies or institutions. More experiments would be necessary to determine if the hypothesis was met as a number of chromosomal abnormalities detected were not always confirmed by other experiments. The hypothesis for chapter 5 was: Can aneuploidy be detected in coelomic fluid using a-CGH? The possibility of using WGA and a-CGH on coelomic fluid was tested as this could be used as an early form of prenatal diagnosis. Coelomic fluid was collected between the 5th and 11th week of pregnancy from women undergoing termination of pregnancy. MDA and GenomePlex were used to amplify the DNA prior to a-CGH analysis. Both genomic (high resolution) and constitutional (low resolution) arrays were tested. The results showed that aneuploidy can be detected by a-CGH. BlueGnome Cytochip slides produced the best results. A triploid sample was detected as normal. The hypothesis was met and even higher resolution could be achieved with the use of GenomePlex and BlueGnome Cytochip arrays. WGA may be very important for downstream genetic tests when the DNA is from very low quality and quantity. Further optimisation of the technique is needed in order to achieve similar results to those of good quality genomic DNA. Arrays from different companies or institutions may produce very different results. In conclusion, the results showed that WGA can benefit PGD and PND, and a-CGH gives great potential to PGS and coelomic fluid diagnosis