Application of a Naïve Bayes Classifier to Assign Polyadenylation Sites from 3\u27 End Deep Sequencing Data: A Dissertation

Cleavage and polyadenylation of a precursor mRNA is important for transcription termination, mRNA stability, and regulation of gene expression. This process is directed by a multitude of protein factors and cis elements in the pre-mRNA sequence surrounding the cleavage and polyadenylation site. Importantly, the location of the cleavage and polyadenylation site helps define the 3’ untranslated region of a transcript, which is important for regulation by microRNAs and RNA binding proteins. Additionally, these sites have generally been poorly annotated. To identify 3’ ends, many techniques utilize an oligo-dT primer to construct deep sequencing libraries. However, this approach can lead to identification of artifactual polyadenylation sites due to internal priming in homopolymeric stretches of adenines. Previously, simple heuristic filters relying on the number of adenines in the genomic sequence downstream of a putative polyadenylation site have been used to remove these sites of internal priming. However, these simple filters may not remove all sites of internal priming and may also exclude true polyadenylation sites. Therefore, I developed a naïve Bayes classifier to identify putative sites from oligo-dT primed 3’ end deep sequencing as true or false/internally primed. Notably, this algorithm uses a combination of sequence elements to distinguish between true and false sites. Finally, the resulting algorithm is highly accurate in multiple model systems and facilitates identification of novel polyadenylation sites

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice

Pathogenic variants i

Nitrous oxide emissions from a peatbog after 13 years of experimental nitrogen deposition

Nitrogen deposition was experimentally increased on a Scottish peatbog over a period of 13 years (2002–2015). Nitrogen was applied in three forms, NH3 gas, NH4Cl solution, and NaNO3 solution, at rates ranging from 8 (ambient) to 64 kg N ha−1 yr−1, and higher near the NH3 fumigation source. An automated system was used to apply the nitrogen, such that the deposition was realistic in terms of rates and high frequency of deposition events. We measured the response of nitrous oxide (N2O) flux to the increased nitrogen input. Prior expectations, based on the IPCC default emission factor, were that 1 % of the added nitrogen would be emitted as N2O. In the plots treated with NH4+ and NO3− solution, no response was seen, and there was a tendency for N2O fluxes to be reduced by additional nitrogen, though this was not significant. Areas subjected to high NH3 emitted more N2O than expected, up to 8.5 % of the added nitrogen. Differences in the response are related to the impact of the nitrogen treatments on the vegetation. In the NH4+ and NO3− treatments, all the additional nitrogen is effectively immobilised in the vegetation and top 10 cm of peat. In the NH3 treatment, much of the vegetation was killed off by high doses of NH3, and the nitrogen was presumably more available to denitrifying bacteria. The design of the wet and dry experimental treatments meant that they differed in statistical power, and we are less likely to detect an effect of the NH4+ and NO3− treatments, though they avoid issues of pseudo-replication

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice

KMT5B gene; Neurodevelopment; MiceGen KMT5B; Neurodesenvolupament; RatolinsGen KMT5B; Neurodesarrollo; RatonesPathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.This work was supported by LB692 Nebraska Tobacco Settlement Biomedical Research Development Program (to H.A.F.S.); The Simons Foundation Autism Research Initiative–Bridge to Independence Award SFARI 381192 (to H.A.F.S.); The A*STAR, Singapore, IAF-PP Program H17/01/a0/004 (to C.Y.L.); The Wong Boon Hock Society research program Yong Loo Lin School of Medicine (to Z.X.C.); NIH training grant 2T32GM008638-25 (L.B.); The Intramural Research Program of the National Human Genome Research Institute (to W.G.); The National Center for Advancing Translational Sciences of the NIH award number TL1TR001880 (to S.E.S.); The Eunice Kennedy Shriver National Institute of Child Health and Human Development award number HD009003-01 (to S.E.S.); Institute for Translational Medicine and Therapeutics of the Perelman School of Medicine at the University of Pennsylvania (to S.E.S.); and Swiss National Science Foundation (SNSF) grant 320020_179547 and funds from the University of Zurich Research Priority Programs (URPP) AdaBD: Adaptive Brain Circuits in Developments (to A.Rau.). F.J.K. was funded by the Deutsche Forschungsgemeinschaft grant number FOR 2488. In silico modeling was supported by the Spanish Ministerio de Ciencia e Innovación grant number PID2019-111217RB-I00 (to X.d.l.C.). This study used data from the DDD study. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003). This study makes use of DECIPHER (www.deciphergenomics.org), which is funded by Wellcome (grant number 223718/Z/21/Z). See Nature PMID: 25533962 or www.ddduk.org/access.html for full acknowledgement

The AllWISE Motion Survey, Part 2

We use the AllWISE Data Release to continue our search for WISE-detected motions. In this paper, we publish another 27,846 motion objects, bringing the total number to 48,000 when objects found during our original AllWISE motion survey are included. We use this list, along with the lists of confirmed WISE-based motion objects from the recent papers by Luhman and by Schneider et al. and candidate motion objects from the recent paper by Gagne et al. to search for widely separated, common-proper-motion systems. We identify 1,039 such candidate systems. All 48,000 objects are further analyzed using color-color and color-mag plots to provide possible characterizations prior to spectroscopic follow-up. We present spectra of 172 of these, supplemented with new spectra of 23 comparison objects from the literature, and provide classifications and physical interpretations of interesting sources. Highlights include: (1) the identification of three G/K dwarfs that can be used as standard candles to study clumpiness and grain size in nearby molecular clouds because these objects are currently moving behind the clouds, (2) the confirmation/discovery of several M, L, and T dwarfs and one white dwarf whose spectrophotometric distance estimates place them 5-20 pc from the Sun, (3) the suggestion that the Na 'D' line be used as a diagnostic tool for interpreting and classifying metal-poor late-M and L dwarfs, (4) the recognition of a triple system including a carbon dwarf and late-M subdwarf, for which model fits of the late-M subdwarf (giving [Fe/H] ~ -1.0) provide a measured metallicity for the carbon star, and (5) a possible 24-pc-distant K5 dwarf + peculiar red L5 system with an apparent physical separation of 0.1 pc.Comment: 62 pages with 80 figures, accepted for publication in The Astrophysical Journal Supplement Series, 23 Mar 2016; second version fixes a few small typos and corrects the footnotes for Table

Capsular Typing Method for Streptococcus agalactiae Using Whole-Genome Sequence Data.

Group B streptococcus (GBS) capsular serotypes are major determinants of virulence and affect potential vaccine coverage. Here we report a whole-genome-sequencing-based method for GBS serotype assignment. This method shows strong agreement (kappa of 0.92) with conventional methods and increased serotype assignment (100%) to all 10 capsular types

Main-Belt Comet P/2012 T1 (PANSTARRS)

We present initial results from observations and numerical analyses aimed at characterizing main-belt comet P/2012 T1 (PANSTARRS). Optical monitoring observations were made between October 2012 and February 2013 using the University of Hawaii 2.2 m telescope, the Keck I telescope, the Baade and Clay Magellan telescopes, Faulkes Telescope South, the Perkins Telescope at Lowell Observatory, and the Southern Astrophysical Research (SOAR) telescope. The object's intrinsic brightness approximately doubles from the time of its discovery in early October until mid-November and then decreases by ~60% between late December and early February, similar to photometric behavior exhibited by several other main-belt comets and unlike that exhibited by disrupted asteroid (596) Scheila. We also used Keck to conduct spectroscopic searches for CN emission as well as absorption at 0.7 microns that could indicate the presence of hydrated minerals, finding an upper limit CN production rate of QCN<1.5x10^23 mol/s, from which we infer a water production rate of QH2O<5x10^25 mol/s, and no evidence of the presence of hydrated minerals. Numerical simulations indicate that P/2012 T1 is largely dynamically stable for >100 Myr and is unlikely to be a recently implanted interloper from the outer solar system, while a search for potential asteroid family associations reveal that it is dynamically linked to the ~155 Myr-old Lixiaohua asteroid family.Comment: 15 pages, 4 figures, accepted for publication in ApJ Letter

Recombination-Mediated Host Adaptation by Avian Staphylococcus aureus

Staphylococcus aureus are globally disseminated among farmed chickens causing skeletal muscle infections, dermatitis, and septicaemia. The emergence of poultry-associated lineages has involved zoonotic transmission from humans to chickens but questions remain about the specific adaptations that promote proliferation of chicken pathogens. We characterized genetic variation in a population of genome-sequenced S. aureus isolates of poultry and humanorigin. Genealogical analysis identified a dominant poultry-associated sequence cluster within the CC5 clonal complex. Poultry and human CC5 isolates were significantly distinct from each other and more recombination events were detected in the poultry isolates. We identified 44 recombination events in 33 genes along the branch extending to the poultry-specific CC5 cluster, and 47 genes were found more often in CC5 poultry isolates compared with those from humans. Many of these gene sequences were common in chicken isolates from other clonal complexes suggesting horizontal gene transfer among poultry associated lineages. Consistent with functional predictions for putative poultry-associated genes, poultry isolates showed enhanced growth at 42 degrees C and greater erythrocyte lysis on chicken blood agar in comparison with human isolates. By combining phenotype information with evolutionary analyses of staphylococcal genomes, we provide evidence of adaptation, following a human-to-poultry host transition. This has important implications for the emergence and dissemination of new pathogenic clones associated with modern agriculture.Peer reviewe

Accuracy of potential diagnostic indicators for coeliac disease:a systematic review protocol

INTRODUCTION: Coeliac disease (CD) is a systemic immune-mediated disorder triggered by gluten in genetically predisposed individuals. CD is diagnosed using a combination of serology tests and endoscopic biopsy of the small intestine. However, because of non-specific symptoms and heterogeneous clinical presentation, diagnosing CD is challenging. Early detection of CD through improved case-finding strategies can improve the response to a gluten-free diet, patients' quality of life and potentially reduce the risk of complications. However, there is a lack of consensus in which groups may benefit from active case-finding. METHODS AND ANALYSIS: We will perform a systematic review to determine the accuracy of diagnostic indicators (such as symptoms and risk factors) for CD in adults and children, and thus can help identify patients who should be offered CD testing. MEDLINE, Embase, Cochrane Library and Web of Science will be searched from 1997 until 2020. Screening will be performed in duplicate. Data extraction will be performed by one and checked by a second reviewer. Disagreements will be resolved through discussion or referral to a third reviewer. We will produce a narrative summary of identified prediction models. Studies, where 2×2 data can be extracted or reconstructed, will be treated as diagnostic accuracy studies, that is, the diagnostic indicators are the index tests and CD serology and/or biopsy is the reference standard. For each diagnostic indicator, we will perform a bivariate random-effects meta-analysis of the sensitivity and specificity. ETHICS AND DISSEMINATION: Results will be reported in peer-reviewed journals, academic and public presentations and social media. We will convene an implementation panel to advise on the optimum strategy for enhanced dissemination. We will discuss findings with Coeliac UK to help with dissemination to patients. Ethical approval is not applicable, as this is a systematic review and no research participants will be involved. PROSPERO REGISTRATION NUMBER: CRD42020170766

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae

Metagenomic sequencing of fecal DNA can usefully characterise an individual’s intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goa