Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets. This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized. The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set. In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained. / QC20100907

Mineralization of soft tissues can cause significantly increased morbidity and mortality. The mechanism for this process is still unknown; however, patients with chronic kidney disease (CKD) are at high risk of developing vascular calcifications. Coronary artery calcification occurs faster in CKD patients undergoing dialysis in comparison with the general population. The pathological process of vascular calcification is the leading cause of death in patients with CKD.   Upper zone of growth plate and cartilage matrix associated protein (UCMA) is a novel vitamin-K dependent (VKD) protein expressed in bone and the vascular system. The UCMA protein contains 15 γ-carboxyglutamic acid (Gla) residues in its 138 residue sequence which is the highest ratio between the number of Gla-residues and the size of the mature protein found in any protein so far. These Gla-residues form a domain that gives unique calcium binding properties for UCMA with high affinity for calcium phosphate crystals (i.e., hydroxyapatite). Even though the function of UCMA remains to be elucidated, it has been speculated that UCMA inhibits calcification of soft tissues and could therefore have a protective function against vascular calcification. Any mutations in the gene coding for UCMA might lead to a diminished function or defective protein.   The aim of this study was to determine whether the gene encoding UCMA in patients with the most progressed stage of CKD (stage 5 CKD) contained any mutations. This was accomplished by performing a full re-sequencing of all five exons with dideoxy sequencing in 16 patients with stage 5 CKD on heamodialysis. If any mutations were discovered, pyrosequencing would be performed on 98 healthy control individuals. This would help to determine if the mutation was exclusive for the patients or existed in the general population as well.   Genomic DNA was extracted from whole blood originating from 16 patients with CKD on haemodialysis. Each of UCMAs five exons were amplified with PCR and the results were visualized using gel electrophoresis. Each exon was re-sequenced and pyrosequencing was performed on 98 healthy control samples. The acquired results were compared with the sequence of the UCMA gene identified at NCBI-GenBank (NCBI, build 37.2, NM_145314.1, Gene ID: 221044) and the Ensemble genome browser (ENSG00000165623). In addition, the frequencies of each SNP were calculated and compared with a study at the Ensemble database originating from the 1000 genomes project (1000GENOMES:low_coverage: CEU).   Because the population of our study group was too small to yield appropriate power for statistical calculations, no definite conclusions could be drawn from the acquired results. Nevertheless, this is the first patient group with CKD ever studied and should thus be regarded as a pilot study due to the limited size. However, no indication was found that UCMA had major defects in the investigated patients. Instead, a heterozygous transversion mutation was found in SNP rs4750328, indicating that the site of this SNP is subject to other modifications. Furthermore, a novel SNP was discovered which has not been described in other populations to our knowledge. The novel SNP is non-synonymous (i.e., causes an amino acid exchange) and located at the carboxyl-terminal of the protein. A serine is incorporated instead of threonine giving a 138Thr>Ser change since the last ACC codon in exon 5 (adjacent to the stop codon) is altered to an AGC codon. The UCMA 138Thr>Ser polymorphism was submitted to the dbSNP database and has been assigned the accession number ss283927876, which will be publicly available upon the release of the next dbSNP Build, B134. In order to determine the physiological significance of the discovered SNP, functional studies are required on both the wild-type and mutated UCMA variants.

Transcriptome analysis based on deep expressed sequence tag (EST) sequencing allows quantitative comparisons of gene expression across multiple species. Using pyrosequencing, we generated over 7 million ESTs from four stages of developing seeds of Ricinus communis, Brassica napus, Euonymus alatus and Tropaeolum majus, which differ in their storage tissue for oil, their ability to photosynthesize and in the structure and content of their triacylglycerols (TAG). The larger number of ESTs in these 16 datasets provided reliable estimates of the expression of acyltransferases and other enzymes expressed at low levels. Analysis of EST levels from these oilseeds revealed both conserved and distinct species-specific expression patterns for genes involved in the synthesis of glycerolipids and their precursors. Independent of the species and tissue type, ESTs for core fatty acid synthesis enzymes maintained a conserved stoichiometry and a strong correlation in temporal profiles throughout seed development. However, ESTs associated with non-plastid enzymes of oil biosynthesis displayed dissimilar temporal patterns indicative of different regulation. The EST levels for several genes potentially involved in accumulation of unusual TAG structures were distinct. Comparison of expression of members from multi-gene families allowed the identification of specific isoforms with conserved function in oil biosynthesis. In all four oilseeds, ESTs for Rubisco were present, suggesting its possible role in carbon metabolism, irrespective of light availability. Together, these data provide a resource for use in comparative and functional genomics of diverse oilseeds. Expression data for more than 350 genes encoding enzymes and proteins involved in lipid metabolism are available at the 'ARALIP' website ().

No description available.

Tardive dyskinesia (TD) is a serious side effect of certain antipsychotic medications that are used to treat schizophrenia (SCZ) and other mental illnesses. The methylation status of the insulin receptor substrate 1 (IRS1) gene is reportedly associated with SCZ; however, no study, to the best of the authors' knowledge, has focused on the quantitative DNA methylation levels of the IRS1 gene using pyrosequencing in SCZ with or without TD. The present study aimed to quantify DNA methylation levels of 4 CpG sites in the IRS1 gene using a Chinese sample including SCZ patients with TD and without TD (NTD) and healthy controls (HCs). The general linear model (GLM) was used to detect DNA methylation levels among the 3 proposed groups (TD vs. NTD vs. HC). Mean DNA methylation levels of 4 CpG sites demonstrated normal distribution. Pearson's correlation analysis did not reveal any significant correlations between the DNA methylation levels of the 4 CpG sites and the severity of SCZ. GLM revealed significant differences between the 3 groups for CpG site 1 and the average of the 4 CpG sites (P=0.0001 and P=0.0126, respectively). Furthermore, the TD, NTD and TD + NTD groups demonstrated lower methylation levels in CpG site 1 (P=0.0003, P<0.0001 and P<0.0001, respectively) and the average of 4 CpG sites (P=0.0176, P=0.0063 and P=0.003, respectively) compared with the HC group. The results revealed that both NTD and TD patients had significantly decreased DNA methylation levels compared with healthy controls, which indicated a significant association between the DNA methylation levels of the IRS1 gene with SCZ and TD.

This thesis includes two studies: The first examined patterns of neutral genetic diversity within Bromus tectorum L. across the IMW region, and uses patterns of microsatellite (SSR) genotype distribution to make inferences about the respective roles of adaptively significant genetic variation, adaptive phenotypic plasticity, and facultative outcrossing in the ongoing invasion and recent range expansion of B. tectorum. It has been previously demonstrated that, due to extremely low outcrossing rates, it is possible to characterize individual genotypes of this species using four SSR loci. We sampled 20 individuals from each of 96 B. tectorum populations (classified by region and habitat) from throughout the IMW and used these SSR markers to characterize each individual. We found 131 four-locus SSR genotypes; however, the 14 most common genotypes collectively accounted for 79.2% of the individuals sampled. Individuals with certain SSR genotypes sorted strongly into warm or salt desert habitats (stringent habitats) and flowered earlier than individuals with genotypes from more mesic habitats, providing evidence of adaptively significant genetic variation associated with these genotypes. Other SSR genotypes were found across a wide range of habitats though they tended to be less prevalent in stringent habitats, providing evidence that adaptive phenotypic plasticity may be important for the distribution of some common genotypes. We observed very few heterozygous individuals, consistent with the highly inbreeding reproductive strategy of B. tectorum. Because specialist genotypes dominating recently invaded areas within the IMW region contained unique alleles, they are not likely to have resulted from recombination, leading us to doubt the role of facultative outcrossing as a significant mechanism facilitating the current range expansion of B. tectorum in the IMW.Previous research investigating the population and ecological genetics of Bromus tectorum L. in the North American invaded range has relied on either allozyme or microsatellite (SSR) genetic analyses, both of which have proven to have shortcomings. In order to overcome the issues associated with these other marker types, in the second study of this thesis we developed single nucleotide polymorphism (SNP) markers for B. tectorum by 1) obtaining normalized cDNA, 2) sequencing normalized cDNA using 454 sequencing, 3) aligning resultant contigs and looking for SNPs, 4) designing assays for SNP validation and genotyping using KASPar, 5) converting working KASPar assays for use with the Fluidigm EP1 platform using the 96.96 Dynamic ArrayTM IFC. Sequencing resulted in 1258041 reads, which assembled into 65486 contigs (20782 large contigs exceeding 500 base pairs). Using selection criteria of at least 10x coverage and 30% of the minor allele, 3333 putative SNPs were identified. We developed KASP assays for 255 putative SNPs, which resulted in 101 working polymorphic assays. Ninety-six assays were then successfully converted for use with KASP on the Fluidigm EP1 genotyping platform using 96.96 dynamic arrays.

The size and complexity of the oat genomes (Avena L., x = 7) have made genetic studies, including the discovery of molecular markers, difficult. Recent attention to these species has resulted in the development of many DArT -based markers in the tetraploid A. magna Murphy et Terrill (2n = 28, CCDD genomes), along with numerous RFLP's, SSR's, DArT's, and EST-based SNPs in hexaploid A. sativa L. (2n = 42, AACCDD). Here we report the first SNP markers for tetraploid oat based on genome reduction and high-throughput pyrosequencing in two inbred lines of A. magna: A-169 (wild) and Ba 13-13 (domesticated). Initially, the genomes were reduced using restriction digests with EcoRI and BfaI and sequenced to produce 706,426 reads for both genotypes that were subsequently assembled into 57,048 contigs with an average read length of 345 bp. Comparisons of the contigs between the two lines resulted in the detection of 31,304 in silico SNPs. High Resolution Melt (HRM) and KASPar assays were used to validate 1,108 of these in silico SNPs across a panel of diploid, tetraploid, and hexaploid oats. Of the assays, 119 were validated using HRM and 384 using KASPar genotyping in the Fluidigm EP1 system. Both sets of assays were then mapped on a population of 117 F2:8 recombinant inbred lines (RILs) developed from the A-169 x Ba 13-13 cross. A map of the A. magna genome was then constructed. The markers and map provide a new set of genomic tools for tetraploid and hexaploid oat breeding and allow for tracking of genes controlling traits of economic importance and other interesting genes through the evolution of Avena.

From acid seeps and deep-sea thermal vents to glacial ice and hypersaline lakes, extreme environments contain relatively simplified communities consisting of extremophiles that have evolved to survive and thrive under adverse abiotic conditions. In more neutral environments, microorganisms use dormancy as a common life history strategy to weather temporal fluctuations of resources or stresses until more 'optimal' conditions are present. It is unclear if dormancy is an essential survival mechanism for microorganisms in extreme environments; however, recent studies suggest that extreme environments may create stable conditions for extremophiles to the extent that dormancy is of less ecological importance. Using lake salinity levels as measurements of "extreme," we evaluated the dormancy of bacterial and archaeal phyla and lake chemistry in five hypersaline and five freshwater lakes across the western United States. Dormancy was calculated using targeted metagenomics to analyze 16S rDNA and rRNA tag sequences. It was hypothesized that bacteria and archaea in hypersaline lake communities would exhibit lower levels dormancy than bacterial and archaeal communities in geologically similar freshwater lake controls. It was also hypothesized that microbial dormancy would decrease as the dominant extreme environmental variable increased in the lakes. As hypothesized, overall dormancy decreased at least 2-fold in hypersaline compared to freshwater lakes for both bacteria and archaea. Of the predominant phyla and subclasses, Firmicutes, Bacteroidetes, and Gammaproteobacteria each demonstrated at least a seven-fold decrease in dormancy in hypersaline lakes compared to freshwater lakes. Specifically, species within the genus Clostridium were responsible for 85% of the dormancy observed in the phylum Firmicutes. Also as hypothesized, microbial dormancy decreased as salinity increased in the lakes. Lower dormancy in hypersaline lakes correlated with increasing salinity while lower dormancy in freshwater lakes correlated with increasing total phosphorus levels. These results suggest that dormancy is a less common life history strategy for microorganisms in extreme environments; it is proposed that this is due to the relatively stable environment in hypersaline lakes and the reduced number of available microbial niches. These results also suggest that the dominant extreme stress (i.e., salinity) may override other driving factors in an environment to ultimately determine microbial community composition, diversity and richness.

Multiple mechanisms control bacterial community structure but two in particular, the "legacy" of past environmental conditions, and the "priming" of bacteria to respond to seasonal or reoccurring fluctuations in resources, have the potential to determine both bacterial communities, as well as, temporal shifts in active bacterial taxa. To begin to evaluate the legacy effects of resources on microbial communities, we added four limiting resources annually (i.e., water only; C-mannitol + water; N-NH4NO3 + water; and C, N + water) and measured shifts in bacterial community composition after seven years in a cold desert ecosystem in the McMurdo Dry Valleys, Antarctica. Further, to investigate the ecological significance of priming, we conducted a series of stable isotope probing experiments (i.e., 18O-DNA SIP with 18O-labeled water, 13C-DNA SIP with 13C-labeled mannitol, 15N-DNA with 15N- NH4NO3, and a combined C and N SIP) and characterized the responding (i.e., isotopically labeled) and seed bank (i.e., unlabeled) bacterial communities. We performed each of the SIPs in soil microcosms corresponding to a single resource manipulation (e.g., 13C-labeled mannitol in C addition soils). We hypothesized that all long-term additions of nutrients and water will lead to a distinct bacterial community—a legacy effect due to the nutrient and water impoverished state of Antarctica soils. We also hypothesized that the stronger the legacy effects demonstrated by a specific community the more adapted or primed bacterial species will be to take advantage of the resource and respond. As hypothesized, resource additions created distinct bacterial legacy but to different degrees among the treatments. The extent of the resource legacy effects was greatest in the CN, intermediate in water and N, and lowest in C communities relative to the control communities, suggesting that C induced changes in communities were intensified by tandem N additions and that water alone created a more distinct legacy than water and C additions combined. Contrary to our hypothesis, the stronger the legacy effects, the less adapted or primed the community was to take advantage of resource additions. For example, the CN treatment that induced the greatest effect on bacterial communities had the lowest number of species (20.9%) in common between the responding and seed bank communities. This inverse relationship may be due to only two species (i.e., Arthrobacter, Actinobacteria and Massilia, Betaproteobacteria) really being primed to take advantage of CN and these species constituting over 75% of the seed bank community. Water, N, and C additions had similar levels of priming with 38.4%, 41.4%, and 36.3% of the responding species being present in the seed bank community, respectively. But of these three treatments, only the priming with water resulted in a unique responding community, suggesting that water, a universal bacterial resource, was enough to prime bacteria. Furthermore, water generates the most diverse responding community of all the resources with stemming from all of the fourteen dominant phyla. We did find patterns of ecological coherence among the responders, especially in the major responders (i.e., responders that increased in relative recovery by at least ten-fold). These responders were predominantly found in only three phyla (i.e., Actinobacteria, Bacteriodetes, and Gammaproteobacteria) regardless of resource addition. Alternatively minor responders (i.e., responders that increased in relative recovery at least two-fold) were contained in fourteen different phyla with specific taxa stimulated by CN (i.e., Betaproteobacteria) and N and water (i.e., Deltaproteobacteria). Further, resource additions elicited responses from 37% of bacterial species with species specializing on a specific resource (e.g., Chloroflexi) or being a generalist (e.g., Planctomycetes and Gammaproteobacteria). Our results offer the first direct links between legacy and priming effects on bacterial community composition and demonstrate that these mechanisms are not always complimentary leading to the formation of similar communities but may both be essential to maintain the high levels of bacterial diversity. Further, all resources produced elicited responders that were either specialists of generalists demonstrating that even bacteria in the extreme environment of Antarctica respond to pulses of resources.

The potential value of the Y chromosome for forensic applications has been recognized for some time with the current work dedicated to Short Tandem Repeat analysis and Single Nucleotide Polymorphism (SNP) discovery. This study examined the ability of two different SNP analysis methods to determine if they could be utilized in forensic applications and ultimately be developed into an established system for Y chromosome SNP analysis. This study examined two principle SNP analysis systems: single base extension and Pyrosequencing. Pyrosequencing was determined to be superior to single base extension, due to the wealth of information provided with sequencing and the flexibility of designing primers for analysis. Using Pyrosequencing, 50 Y chromosome loci were examined and the minimum loci required for maximum diversity for the development of a Y chromosome SNP analysis system were chosen. Thirteen loci were selected based on their ability to discriminate 60 different individuals from three different racial groups into 15 different haplogroups. The Y chromosome SNP analysis system developed utilized nested PCR for the amplification of all 13 loci. Then they were sequenced as groups, ranging from one to three loci, in a single reaction. The Y chromosome SNP analysis system developed here has the potential for forensic application since it has shown to be successful in the analysis of blood, buccal swabs, semen, and saliva, works with as little as 5 pg of starting DNA material, and will amplify only male DNA in the presence of male/female mixtures in which the female portion of the sample overwhelmed the male portion 30,000 to 1.