The role of individual soil microorganisms changes over the course of a plant's life - microorganisms that have no discernable role at one developmental stage may affect the plant later in its growth. Traditional analysis of the soil microbiome, which has focused principally on the relative abundances (RA) of individual organisms, may be incomplete, as underlying differences in population size cannot be addressed. We conducted a metagenomic analysis of soil microorganisms from various maize (Zea mays L.) fields at two depths, accompanied by crop yield components, to provide insight into influences of edaphic microbes on maize productivity under commercial maize production systems in Missouri. This study assesses the influence of fungi and bacteria, not only in terms of RA, but also in their estimated absolute abundances (EAA), derived by combining the results of Illumina HiSeq sequencing data and phospholipid fatty acid abundance data. Significant interactions were identified between maize yield components and soil microbes at critical developmental states. Most interactions between fungi and yield components were negative, with notable exceptions. Bacterial interactions were more complex, with most interactions during early ear development identified as positive, and most interactions during tasseling identified as negative. In addition to the effects that microbial populations have on yield, plant populations reciprocally changed the microbial community. Plant developmental state was the greatest predictor of bacteria, with the microbial communities present during the active growing season being most similar to each other, whereas the preplant microbiome and post-reproductive microbiome being most similar to each other. Fungal communities were primarily dependent on location.

>Magister Scientiae - MSc / Molecular methods were used to investigate the microbial diversity and community structure of ammonia-oxidizing bacteria (AOB) associated with the roots of the Proteaceae plant family. The identification of ammonia oxidizing bacteria in this ecosystem is of particular interest since Proteaceae are adapted to acidic, low nutrient (e.g. nitrogen) soils. The ammonia monooxygenase operon was used as a molecular marker to identify ammonia-oxidizing bacteria associated with the proteoid roots of the three Proteaceae members and compared to non-plant associated soil. PCR amplification using primer sets targeting the ammonia monooxygenase gene (amoA subunits) were used to construct a clone library. Sequence diversity was determined by RFLP analysis of amoA to identify major groups of AOB of the ~-subclass of Proteobacteria in total community DNA, and DNA sequencing and phylogenetic analysis were also applied. DGGE analysis was performed to determine the community structure and distribution of ammonia-oxidizing bacteria in plant-associated and non-plant associated soils. The AOB genotypic diversity was similar in the plant-associated samples and non-plant associated soil. All AOB phylotypes belonged to Nitrosospira species and clustered with Nitrosospira cluster 3. The abundance of the amoA was quantified to be approximately 4.2 x 107 copies/g of dry soil, using a real-time PCR assay. These data suggest that the Nitrosospira species are the dominant phylotypes in that environment. This investigation provides new insights into the relationships between plants and ammonia-oxidizing bacteria in natural Fynbos ecosystems.

With the ability to produce reads with hundreds of kilobases in length, long-read sequencing technology is emerging as a powerful tool to decode complex genetic sequences that are previously inaccessible for short reads. Though the sequencing chemistry and base calling algorithm are being actively developed, the accuracy of the current long-read sequencing is still considerably low, thus limiting its applications. In this dissertation, I present three long read based DNA sequencing methods to overcome the limitation of read accuracy, contribute to a better understanding of Cas9 editing outcomes and mitochondrial DNA heterogeneity, and pave the way for real-time pathogen detection and mutation surveillance. The development of IDMseq enables the single-base-resolution haplotype-resolved quantitative characterization of diverse types of rare variants. IDMseq provides the first quantitative evidence of persistent nonrandom large structural variants following repair of double-strand breaks induced by CRISPR-Cas9 in human ESCs. The development of iMiGseq represents the first mitochondrial DNA sequencing method that provides ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mitochondrial DNA molecule level. iMiGseq uncovers unappreciated levels of heteroplasmic variants in single healthy human oocytes well below the current 1% detection limit, of which numerous variants are deleterious and associated with late-onset mitochondrial disease and cancer. It could comprehensively characterize and haplotype single-nucleotide and structural variants of mitochondrial DNA and their genetic linkage in NARP/Leigh syndrome patient-derived cells. The development of NIRVANA deals with the COVID-19 pandemic. NIRVANA can simultaneously detect SARS-CoV-2 and three co-infecting respiratory viruses, and monitor mutations for up to 96 samples in real time. It provides a promising solution for rapid field-deployable detection and mutation surveillance of pandemic viruses. Taken all together, IDMseq, iMiGseq and NIRVANA utilize the advantage of long reads, overcome the limitation of low accuracy, and facilitate the application of long-read sequencing technologies in multidisciplinary fields.

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.

>Magister Scientiae - MSc / Knowledge of the feeding ecology of an organism helps us to better understand predator-prey relationships and aspects of species biology, ecology and life-history traits. Understanding the feeding ecologies of snakes is challenging because snakes are generally secretive and often difficult to observe when foraging in the wild. Traditionally, studies attempting to quantify the diets of snakes relied on observing direct predation events, dissecting dead specimens, or microscopy of gut and stomach contents to identify prey species. However, investigations using traditional methods can result in an incomplete understanding of prey utilised by particular snakes. Analysis of prey DNA in snake faeces is a useful method to obtain accurate information on diet.

The development of Massively Parallel Sequencing (MPS) has enabled more accurate and less time-consuming DNA sequencing. Although MPS technologies are theoretically applicable to all samples and species, the majority of studies on microorganisms have been conducted on those able to be isolated and cultivated in laboratories. In the field of metagenomics, DNA from uncultivated environmental samples is analyzed. Whole genome sequencing of such complex samples poses difficult computational challenges due to the characteristics of metagenomic data, where one major challenge lies in determining the true origin of high similarity reads. In addition, the short-range information acquired from MPS reveals little about how reads from DNA sequencing fit together. Consequently, producing genome drafts from reads generated by MPS remains difficult. Here, the linked-read sequencing technology DB-Seq has been applied to bacterial samples in order to assess its potential in metagenomics. Specifically, its performance in retaining long-range information in de novo whole genome assembly has been tested. The results obtained in this initial study show great potential of DB-Seq in genome assembly, with significantly more contiguous results than conventional methods generate. / Utvecklingen av Massiv Parallel Sekvensering (MPS) har möjliggjort mer korrekt och mindre tidskrävande DNA sekvensering. Trots att MPS teoretiskt sett kan appliceras på alla provtyper och arter, har majoriteten av de studier som utförts på mikroorganismer varit fokuserade på de som kan isoleras och odlas i laboratorium. Inom ämnet metagenomik analyseras DNA från orörda miljöprover. Helgenomssekvensering av sådana prover ger upphov till komplicerade utmaningar för data-analys, där ett av de största problemen är att bestämma ursprunget av snarlika sekvenseringsresultat. Ytterligare komplikationer uppstår på grund av den data som erhålls från MPS, då denna ej ger information om hur sekvenseringsdata bör placeras i förhållande till varandra. Följdaktligen är det svårt att producera hopsatta genom utifrån MPS-data. I detta projekt har "linked-read"-sekvenseringsteknologin DB-Seq applicerats på bakterieprover för att undersöka metodens potential i metagenomik. Specifikt har metodens förmåga att bibehålla information om ursprungspositionen av sekvenseringsdata testats i de novo sammansättning av genom. De erhållna resultaten i denna förstagångsstudie tyder på stor potential för DB-Seq i genomsammansättning, med signifikant mer sammanhängande resultatsekvenser än vad konventionella metoder uppvisar.

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.

Atriplex hortensis (2n = 2x = 18, 1C genome size ~1.1 gigabases), also known as garden orach, is a highly nutritious, broadleaf annual of the Amaranthaceae-Chenopodiaceae family that has spread from its native Eurasia to other temperate and subtropical environments worldwide. Atriplex is a highly complex and polyphyletic genus of generally halophytic and/or xerophytic plants, some of which have been used as food sources for humans and animals alike. Although there is some literature describing the taxonomy and ecology of orach, there is a lack of genetic and genomic data that would otherwise help elucidate the genetic variation, phylogenetic position, and future potential of this species. Here, we report the assembly of the first highquality, chromosome-scale reference genome for orach cv. ‘Golden’. Sequence data was produced using Oxford Nanopore’s MinION sequencing technology in conjunction with Illumina short-reads and chromatin-contact mapping. Genome assembly was accomplished using the high-noise, single-molecule sequencing assembler, Canu. The genome is enriched for highly repetitive DNA (68%). The Canu assembly combined with the Hi-C chromatin-proximity data yielded a final assembly containing 1,325 scaffolds with a contig N50 of 98.9 Mb and with 94.7% of the assembly represented in the nine largest, chromosome-scale scaffolds. Sixty-eight percent of the genome was classified as highly repetitive DNA, with the most common repetitive elements being Gypsy and Copia-like LTRs. The annotation was completed using MAKER which identified 31,010 gene models and 2,555 tRNA genes. Completeness of the genome was assessed using the Benchmarking Universal Single Copy Orthologs (BUSCO) platform, which quantifies functional gene content using a large core set of highly conserved orthologous genes (COGs). Of the 1,375 plant-specific COGs in the Embryophyta database, 1,330 (96.7%) were identified in the Atriplex assembly. We also report the results of a resequencing panel consisting of 21 accessions which illustrates a high degree of genetic similarity among cultivars and wild material from various locations in North America and Europe. These genome resources provide vital information to better understand orach and facilitate future study and comparison.

Metagenomics is a cultivation-independent approach for obtaining the genomic composition of microbial communities. Microbial communities are ubiquitous in nature. Microbes which are associated with the human body play important roles in human health and disease. These roles span from protecting us against infections from other bacteria, to being the causes of these diseases. A deeper understanding of these communities and how they function inside our bodies allows for advancements in treatments and preventions for these diseases. Recent developments in metagenomics have been driven by the emergence of Next-Generation Sequencing technologies and Third-Generation Sequencing technologies that have enabled cost-effective DNA sequencing and the generation of large volumes of genomic data. These technologies have allowed for the introduction of hybrid DNA assembly techniques to recover the genomes of the constituent microbes. While Next-Generation Sequencing technologies use paired-end sequencing reads from DNA fragments into short reads and have a relatively lower error rate, Third-Generation Sequencing technologies use much longer DNA fragments to generate longer reads, bringing contigs together for larger scaffolds with a higher error rate. Hybrid assemblers leverage both short and long read sequencing technologies and can be a critical step in the advancements of metagenomics, combining these technologies to allow for longer assemblies of DNA with lower error rates. We evaluate the strengths and weaknesses of the hybrid assembly framework using several state-of-the-art assemblers and simulated human microbiome datasets. Our work provides insights into metagenomic assembly and genome recovery, an important step towards a deeper understanding of the microbial communities that influence our well-being.

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