Using bioinformatics to analyze the role of microbial taxa in complex ecosystems

Microbes are abundant on earth and play a crucial role in the environment they inhabit. Before the dawn of metagenomics, the study of the effect of microorganisms on their environment was limited due to use of low throughput techniques that could only examine single organisms or a few at a time. Metagenomics is a fast growing field of science that permits investigation of microbes by directly extracting DNA from the environment. A lot of environments, ranging in complexity from the ocean to acid mines, from wastewater communities to the human body have been targeted by metagenomics studies, and these studies generate tremendous amounts of data and newer and more efficient bioinformatic tools and methods are needed to interpret this complex data. In this dissertation we used bioinformatic tools to enrich our understanding of the role that microorganisms play within some important but understudied microbial environments. In Chapter # 1, we report an increased microbial richness associated with colorectal cancer. This is an important finding that could lead to the development of diagnostic methods to identify individuals at high risk of developing colorectal cancer and this early detection could help devise preventive strategies. In Chapter # 2 we discuss a batch-effect we discovered in our colorectal cancer project and how filtering out the batch-effect helped us in revealing the true biological signal. In Chapter #3 we report results of a metagenomic survey where we analyzed the pyrosequences obtained from a wastewater community. In Chapter # 4 of this dissertation we perform a systematic comparison of some of the methods used in taxonomic profiling of microbial communities and show how the choice of method can have an effect on a community’s taxonomic profile. Overall, this dissertation demonstrates the value of using bioinformatic tools during the course of analysis of complex communities, in not only filtering out artifacts and in choice of analysis pathways but also in discovering important biological effects

Development and comparison of a minimally-invasive model of autologous clot pulmonary embolism in Sprague-Dawley and Copenhagen rats

Background Experimental models of pulmonary embolism (PE) that produce pulmonary hypertension (PH) employ many different methods of inducing acute pulmonary occlusion. Many of these models induce PE with intravenous injection of exogenous impervious objects that may not completely reproduce the physiological properties of autologous thromboembolism. Current literature lacks a simple, well-described rat model of autlogous PE. Objective: Test if moderate-severity autologous PE in Sprague-Dawley (SD) and Copenhagen (Cop) rats can produce persistent PH. Methods blood was withdrawn from the jugular vein, treated with thrombin-Ca++ and re-injected following pretreatment with tranexamic acid. Hemodynamic values, clot weights and biochemical measurements were performed at 1 and 5 days. Results Infusion of clot significantly increased the right ventricular peak systolic pressure to 45-55 mm Hg, followed by normalization within 24 hours in SD rats, and within 5 days in COP rats. Clot lysis was 95% (24 hours) and 97% (5 days) in SD rats and was significantly lower in COP rats (70%, 24 hours; 87% 5 days). Plasma D-dimer was elevated in surgical sham animals and was further increased 8 hours after pulmonary embolism. Neither strain showed a significant increase in bronchoalveolar chemotactic activity, myeloperoxidase activity, leukocyte infiltration, or chemokine accumulation, indicating that there was no significant pulmonary inflammation. Conclusions Both SD and COP rats exhibited near complete fibrinolysis of autologous clot PE within 5 days. Neither strain developed persistent PH. Experimental models of PE designed to induce sustained PH and a robust inflammatory response appear to require significant, persistent pulmonary vascular occlusion

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn's disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1–V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas

Development and comparison of a minimally-invasive model of autologous clot pulmonary embolism in Sprague-Dawley and Copenhagen rats

Abstract Background Experimental models of pulmonary embolism (PE) that produce pulmonary hypertension (PH) employ many different methods of inducing acute pulmonary occlusion. Many of these models induce PE with intravenous injection of exogenous impervious objects that may not completely reproduce the physiological properties of autologous thromboembolism. Current literature lacks a simple, well-described rat model of autlogous PE. Objective: Test if moderate-severity autologous PE in Sprague-Dawley (SD) and Copenhagen (Cop) rats can produce persistent PH. Methods blood was withdrawn from the jugular vein, treated with thrombin-Ca++ and re-injected following pretreatment with tranexamic acid. Hemodynamic values, clot weights and biochemical measurements were performed at 1 and 5 days. Results Infusion of clot significantly increased the right ventricular peak systolic pressure to 45-55 mm Hg, followed by normalization within 24 hours in SD rats, and within 5 days in COP rats. Clot lysis was 95% (24 hours) and 97% (5 days) in SD rats and was significantly lower in COP rats (70%, 24 hours; 87% 5 days). Plasma D-dimer was elevated in surgical sham animals and was further increased 8 hours after pulmonary embolism. Neither strain showed a significant increase in bronchoalveolar chemotactic activity, myeloperoxidase activity, leukocyte infiltration, or chemokine accumulation, indicating that there was no significant pulmonary inflammation. Conclusions Both SD and COP rats exhibited near complete fibrinolysis of autologous clot PE within 5 days. Neither strain developed persistent PH. Experimental models of PE designed to induce sustained PH and a robust inflammatory response appear to require significant, persistent pulmonary vascular occlusion.</p

Molecular Diversity of a North Carolina Wastewater Treatment Plant as Revealed by Pyrosequencing▿ †

We report the results of pyrosequencing of DNA collected from the activated sludge basin of a wastewater treatment plant in Charlotte, NC. Using the 454-FLX technology, we generated 378,601 sequences with an average read length of 250.4 bp. Running the 454 assembly algorithm over our sequences yielded very poor assembly, with only 0.3% of our sequences participating in assembly of significant contigs. Of the 117 contigs greater than 500 bp long that were assembled, the most common annotations were to transposases and hypothetical proteins. Comparing our sequences to known microbial genomes showed nonspecific recruitment, indicating that previously described taxa are only distantly related to the most abundant microbes in this treatment plant. A comparison of proteins generated by translating our sequence set to translations of other sequenced microbiomes shows a distinct metabolic profile for activated sludge with high counts for genes involved in metabolism of aromatic compounds and low counts for genes involved in photosynthesis. Taken together, these data document the substantial levels of microbial diversity within activated sludge and further establish the great utility of pyrosequencing for investigating diversity in complex ecosystems

Assessment of an automated approach for variant interpretation in screening for monogenic disorders: A single‐center study

Abstract Background Automation has been introduced into variant interpretation, but it is not known how automated variant interpretation performs on a stand‐alone basis. The purpose of this study was to evaluate a fully automated computerized approach. Method We reviewed all variants encountered in a set of carrier screening panels over a 1‐year interval. Observed variants with high‐confidence ClinVar interpretations were included in the analysis; those without high‐confidence ClinVar entries were excluded. Results Discrepancy rates between automated interpretations and high‐confidence ClinVar entries were analyzed. Of the variants interpreted as positive (likely pathogenic or pathogenic) based on ClinVar information, 22.6% were classified as negative (variants of uncertain significance, likely benign or benign) variants by the automated method. Of the ClinVar negative variants, 1.7% were classified as positive by the automated software. On a per‐case basis, which accounts for variant frequency, 63.4% of cases with a ClinVar high‐confidence positive variant were classified as negative by the automated method. Conclusion While automation in genetic variant interpretation holds promise, there is still a need for manual review of the output. Additional validation of automated variant interpretation methods should be conducted

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn's disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1–V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas