Enabling high-throughput sequencing data analysis with MOSAIK

Thesis advisor: Gabor T. MarthDuring the last few years, numerous new sequencing technologies have emerged that require tools that can process large amounts of read data quickly and accurately. Regardless of the downstream methods used, reference-guided aligners are at the heart of all next-generation analysis studies. I have developed a general reference-guided aligner, MOSAIK, to support all current sequencing technologies (Roche 454, Illumina, Applied Biosystems SOLiD, Helicos, and Sanger capillary). The calibrated alignment qualities calculated by MOSAIK allow the user to fine-tune the alignment accuracy for a given study. MOSAIK is a highly configurable and easy-to-use suite of alignment tools that is used in hundreds of labs worldwide. MOSAIK is an integral part of our genetic variant discovery pipeline. From SNP and short-INDEL discovery to structural variation discovery, alignment accuracy is an essential requirement and enables our downstream analyses to provide accurate calls. In this thesis, I present three major studies that were formative during the development of MOSAIK and our analysis pipeline. In addition, I present a novel algorithm that identifies mobile element insertions (non-LTR retrotransposons) in the human genome using split-read alignments in MOSAIK. This algorithm has a low false discovery rate (4.4 %) and enabled our group to be the first to determine the number of mobile elements that differentially occur between any two individuals.Thesis (PhD) — Boston College, 2010.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

Professional Reading

The Teaching of Ethics in the Militar

Patterns of MicroRNA Expression in Normal and Early Alzheimer\u27s Disease Human Temporal Cortex: White Matter versus Gray Matter

MicroRNA (miRNA) expression was assessed in human cerebral cortical gray matter (GM) and white matter (WM) in order to provide the first insights into the difference between GM and WM miRNA repertoires across a range of Alzheimer\u27s disease (AD) pathology. RNA was isolated separately from GM and WM portions of superior and middle temporal cerebral cortex (N = 10 elderly females, postmortem interval \u3c 4 h). miRNA profiling experiments were performed using state-of-the-art Exiqon© LNA-microarrays. A subset of miRNAs that appeared to be strongly expressed according to the microarrays did not appear to be conventional miRNAs according to Northern blot analyses. Some well-characterized miRNAs were substantially enriched in WM as expected. However, most of the miRNA expression variability that correlated with the presence of early AD-related pathology was seen in GM. We confirm that downregulation of a set of miRNAs in GM (including several miR-15/107 genes and miR-29 paralogs) correlated strongly with the density of diffuse amyloid plaques detected in adjacent tissue. A few miRNAs were differentially expressed in WM, including miR-212 that is downregulated in AD and miR-424 which is upregulated in AD. The expression of certain miRNAs correlates with other miRNAs across different cases, and particular subsets of miRNAs are coordinately expressed in relation to AD-related pathology. These data support the hypothesis that patterns of miRNA expression in cortical GM may contribute to AD pathogenetically, because the aggregate change in miRNA expression observed early in the disease would be predicted to cause profound changes in gene expression

Anti-Argonaute RIP-Chip Shows that miRNA Transfections Alter Global Patterns of mRNA Recruitment to Microribonucleoprotein Complexes

MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the RIP-Chip assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs

Individual MicroRNAs (miRNAs) Display Distinct mRNA Targeting Rules

MicroRNAs (miRNAs) guide Argonaute (AGO)-containing microribonucleoprotein (miRNP) complexes to target mRNAs.It has been assumed that miRNAs behave similarly to each other with regard to mRNA target recognition. The usual assumptions, which are based on prior studies, are that miRNAs target preferentially sequences in the 3\u27UTR of mRNAs,guided by the 5\u27 seed portion of the miRNAs. Here we isolated AGO- and miRNA-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with anti-AGO antibody. Cells were transfected with miR-107, miR-124,miR-128, miR-320, or a negative control miRNA. Co-IPed RNAs were subjected to downstream high-density Affymetrix Human Gene 1.0 ST microarray analyses using an assay we validated previously-a RIP-Chip experimental design. RIP-Chip data provided a list of mRNAs recruited into the AGO-miRNP in correlation to each miRNA. These experimentally identified miRNA targets were analyzed for complementary six nucleotide seed sequences within the transfected miRNAs. We found that miR-124 targets tended to have sequences in the 3\u27UTR that would be recognized by the 5\u27 seed of miR-124, as described in previous studies. By contrast, miR-107 targets tended to have \u27seed\u27 sequences in the mRNA open reading frame, but not the 3\u27 UTR. Further, mRNA targets of miR-128 and miR-320 are less enriched for 6-mer seed sequences in comparison to miR-107 and miR-124. In sum, our data support the importance of the 5\u27 seed in determining binding characteristics for some miRNAs; however, the binding rules are complex, and individual miRNAs can have distinct sequence determinants that lead to mRNA targeting

The Expression of MicroRNA miR-107 Decreases Early in Alzheimer\u27s Disease and May Accelerate Disease Progression through Regulation of β-Site Amyloid Precursor Protein-Cleaving Enzyme 1

MicroRNAs (miRNAs) are small regulatory RNAs that participate in posttranscriptional gene regulation in a sequence-specific manner. However, little is understood about the role(s) of miRNAs in Alzheimer\u27s disease (AD). We used miRNA expression microarrays on RNA extracted from human brain tissue from the University of Kentucky Alzheimer\u27s Disease Center Brain Bank with near-optimal clinicopathological correlation. Cases were separated into four groups: elderly nondemented with negligible AD-type pathology, nondemented with incipient AD pathology, mild cognitive impairment (MCI) with moderate AD pathology, and AD. Among the AD-related miRNA expression changes, miR-107 was exceptional because miR-107 levels decreased significantly even in patients with the earliest stages of pathology. In situ hybridization with cross-comparison to neuropathology demonstrated that particular cerebral cortical laminas involved by AD pathology exhibit diminished neuronal miR-107 expression. Computational analysis predicted that the 3′-untranslated region (UTR) of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA is targeted multiply by miR-107. From the same RNA material analyzed on miRNA microarrays, mRNA expression profiling was performed using Affymetrix Exon Array microarrays on nondemented, MCI, and AD patients. BACE1 mRNA levels tended to increase as miR-107 levels decreased in the progression of AD. Cell culture reporter assays performed with a subset of the predicted miR-107 binding sites indicate the presence of at least one physiological miR-107 miRNA recognition sequence in the 3′-UTR of BACE1 mRNA. Together, the coordinated application of miRNA profiling, Affymetrix microarrays, new bioinformatics predictions, in situ hybridization, and biochemical validation indicate that miR-107 may be involved in accelerated disease progression through regulation of BACE1

Focus on RNA Isolation: Obtaining RNA for MicroRNA (miRNA) Expression Profiling Analyses of Neural Tissue

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of \u27upstream\u27 variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed

Coal Power Development

For the past several years Alstom Power Inc. (Alstom), a leading world-wide power system manufacturer and supplier, has been in the initial stages of developing an entirely new, ultra-clean, low cost, high efficiency power plant for the global power market. This new power plant concept is based on a hybrid combustion-gasification process utilizing high temperature chemical and thermal looping technology The process consists of the oxidation, reduction, carbonation, and calcination of calcium-based compounds, which chemically react with coal, biomass, or opportunity fuels in two chemical loops and one thermal loop. The chemical and thermal looping technology can be alternatively configured as (i) a combustion-based steam power plant with CO{sub 2} capture, (ii) a hybrid combustion-gasification process producing a syngas for gas turbines or fuel cells, or (iii) an integrated hybrid combustion-gasification process producing hydrogen for gas turbines, fuel cells or other hydrogen based applications while also producing a separate stream of CO{sub 2} for use or sequestration. In its most advanced configuration, this new concept offers the promise to become the technology link from today's Rankine cycle steam power plants to tomorrow's advanced energy plants. The objective of this work is to develop and verify the high temperature chemical and thermal looping process concept at a small-scale pilot facility in order to enable AL to design, construct and demonstrate a pre-commercial, prototype version of this advanced system. In support of this objective, Alstom and DOE started a multi-year program, under this contract. Before the contract started, in a preliminary phase (Phase 0) Alstom funded and built the required small-scale pilot facility (Process Development Unit, PDU) at its Power Plant Laboratories in Windsor, Connecticut. Construction was completed in calendar year 2003. The objective for Phase I was to develop the indirect combustion loop with CO{sub 2} separation, and also syngas production from coal with the calcium sulfide (CaS)/calcium sulfate (CaSO{sub 4}) loop utilizing the PDU facility. The results of Phase I were reported in Reference 1, 'Hybrid Combustion-Gasification Chemical Looping Coal Power Development Technology Development Phase I Report' The objective for Phase II was to develop the carbonate loop--lime (CaO)/calcium carbonate (CaCO{sub 3}) loop, integrate it with the gasification loop from Phase I, and ultimately demonstrate the feasibility of hydrogen production from the combined loops. The results of this program were reported in Reference 3, 'Hybrid Combustion-Gasification Chemical Looping Coal Power Development Technology Development Phase II Report'. The objective of Phase III is to operate the pilot plant to obtain enough engineering information to design a prototype of the commercial Chemical Looping concept. The activities include modifications to the Phase II Chemical Looping PDU, solids transportation studies, control and instrumentation studies and additional cold flow modeling. The deliverable is a report making recommendations for preliminary design guidelines for the prototype plant, results from the pilot plant testing and an update of the commercial plant economic estimates

Association of Phosphate-Containing versus Phosphate-Free Solutions on Ventilator Days in Patients Requiring Continuous Kidney Replacement Therapy

Background and objectives Hypophosphatemia is commonly observed in patients receiving continuous KRT. Patients who develop hypophosphatemia may be at risk of respiratory and neuromuscular dysfunction and therefore subject to prolongation of ventilator support. We evaluated the association of phosphate-containing versus phosphate-free continuous KRT solutions with ventilator dependence in critically ill patients receiving continuous KRT. Design, setting, participants, & measurements Our study was a single-center, retrospective, pre-post cohort study of adult patients receiving continuous KRT and mechanical ventilation during their intensive care unit stay. Zeroinflated negative binomial regression with and without propensity score matching was used to model our primary outcome: ventilator-free days at 28 days. Intensive care unit and hospital lengths of stay as well as hospital mortality were analyzed with a t test or a chi-squared test, as appropriate. Results We identified 992 eligible patients, of whom 649 (65%) received phosphate-containing solutions and 343 (35%) received phosphate-free solutions. In multivariable models, patients receiving phosphate-containing continuous KRT solutions had 12% (95% confidence interval, 0.17 to 0.47) more ventilator-free days at 28 days. Patients exposed to phosphate-containing versus phosphate-free solutions had 17% (95% confidence interval, 20.08 to 20.30) fewer days in the intensive care unit and 20% (95% confidence interval, 2 0.12 to 20.32) fewer days in the hospital. Concordant results were observed for ventilator-free days at 28 days in the propensity score matched analysis. There was no difference in hospital mortality between the groups. Conclusions The use of phosphate-containing versus phosphate-free continuous KRT solutions was independently associated with fewer ventilator days and shorter stay in the intensive care unit

STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME): An extension of the STROBE statement

Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility and clinical outcomes are used as proxies for investigating interactions between external and / or endogenous agents and body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as the STrengthening Reporting of OBservational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE statement implementing nine existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendation