Characterizing Sequencing Artifacts

Next Generation Sequencing (NGS) introduces artifactual variants from library preparation methods and errors, which affects the accuracy of variant calling. Whole Exome Sequencing (WES) data from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database is processed. Comparison of single nucleotide polymorphism (SNP) calls to Genome In a Bottle (GIAB) provides labels that are used to build machine learning (ML) models. The left and right flanking region (LSEQ and RSEQ) of each SNP is extracted. Nucleotide frequency, kmers of size 4 and their counts, largest homopolymer size, largest palindrome size, and largest hairpin loop size were computed and used as features in model building. The Random Forest model had a precision of 98.8%, recall of 87.3%, and accuracy of 90.2%. High scores show the model\u27s ability to correctly identify artifacts from non-artifacts and that the results are exceptionally accurate

Development and analysis of molecular methods for functional metagenomics of the human gut microbiome

Interest in the human microbiome has risen quickly in recent years as the microbes that live in and on our body have been implicated in a growing number of human health and disease states. This interest has been supported by advances in DNA sequencing technology that have allowed us to obtain vast amounts of sequence data, and yet we have difficulty assigning function to many of the gene sequences obtained. As research on the role of these microorganisms continues, there will be an increased need for high-throughput methods that can provide knowledge of microbial gene function. Functional metagenomics is one such method, and it relies on first cloning environmental DNA to generate metagenomic libraries that are maintained in Escherichia coli and second, screening the cloned DNA for particular functions of interest. This powerful function-first method allows for the isolation of genes whose role may not have been predicted using DNA sequence homology. This thesis describes the analysis of techniques used in functional metagenomics research, as well as the development of new strategies to aid in functional screening of metagenomic libraries, particularly those constructed from gut-derived DNA. The work is divided into four data chapters that each explore a distinct aspect of the functional metagenomics approach. The first data chapter describes the evaluation of a pooled strategy for sequencing cosmid clones that were previously isolated in functional screens of metagenomic libraries. Ninety-two large-insert clones were pooled for Illumina-sequencing and the assembled sequence data were evaluated against reference sequence data that were obtained from individual barcoded Illumina sequencing of the same clones. The results indicated that a pooled strategy works well provided that sufficient sequencing depth is obtained and that pooled clones do not share sequence similarity to the extent that would be problematic for assembly of short reads that derive from those clones. The second data chapter is an exploration of possible causes for the known cloning bias of metagenomic libraries, by comparing environmental DNA before cloning to the DNA cloned in the final metagenomic library in E. coli. For a human gut metagenomic library, DNA was sampled and Illumina-sequenced at three different steps during the construction of the library. Analyses of the sequence data showed that there was indeed major bias in the final library, but that the bias was not due to fragmentation of the DNA during the cloning process as has been previously suggested; rather, the data were consistent with alternative hypotheses that suggest bias occurs after the DNA is introduced into E. coli, and analyses provide support for the hypothesis that spurious transcription of foreign DNA in E. coli may be contributing to the bias of libraries. Bias was also examined for a soil metagenomic library using 16S rRNA gene sequencing and though broad phylum-level biases were not as severe as observed for the human gut library, analyses revealed a bias in the relative abundance of individual OTUs. The third data chapter describes efforts to develop Bacteroides thetaiotaomicron (B. theta) VPI-5482 as a surrogate host for screening metagenomic libraries constructed from human gut-derived DNA. In this strategy, metagenomic libraries that have been constructed in E. coli can be transferred to B. theta using triparental conjugation. A member of the Bacteroidetes was chosen to specifically address the likely barrier to gene expression in E. coli of DNA that originates from this phylum. To allow the library to be replicated in B. theta, a B. theta-compatible library cloning vector was constructed, and this vector was used to generate genomic and metagenomic clone libraries. A metagenomic library was successfully screened in B. theta, leading to functional complementation of a B. theta mutant strain unable to grow on chondroitin sulfate as sole carbon source. However, further examination of the complemented clones indicated that the library clone DNA had integrated into the B. theta mutant genome. To address this problem, an alternative method for screening was devised, and although this method demonstrates that screening in B. theta remains feasible, more work is required to optimize the conjugation efficiency and the level of throughput. The fourth and last data chapter is an exploration of the use of transcriptional terminator elements in library cloning vectors, inspired by the results of previous chapters. Two unidirectional transcriptional terminators were added to a copy number-inducible fosmid vector, flanking the cloning site, with the intention of reducing insert-born transcription into the vector backbone. The terminators were tested using a reporter gene to confirm their functionality in this context, and derivative vectors were generated for future testing of whether or in what contexts terminators may help alleviate cloning bias in metagenomic libraries. The work described in this thesis contributes to method advancement for functional metagenomics through the analysis of a cost-effective strategy for sequencing library clones, the examination of potential causes of sequence bias in metagenomic libraries, the development of a surrogate host for more productive functional screening, and the consideration of vector elements that may improve metagenomic library stability in E. coli

Physico-chemical properties of fourteen popular cocoa bean varieties in Dongnai-highland Vietnam

Cocoa (Theobroma cacao L.) is a major, crucial economic, global crop and has been maintained several nutritional benefits. The exporting volume of Vietnamese cocoa bean is increasing in the world cocoa trade. The beans of fourteen popular cocoa varieties that are commonly cultivated in Trang Bom (Dong Nai, Vietnam) were characterized for their physico-chemical qualifications on the background of post-fermented cocoa beans and dehydration in the region of Trang Bom-Dong Nai. The physical properties were determined based on mass ratio and bean’s dimension (lengh, width and thickness) as well as chemical (proximate) composition (total crude protein, ash, moisture, and lipid). These values were analysed using AOAC methods. The largest-size beans were found for the TD8 variety (1.5 g of mass, 25.02 mm of length, 14.28 mm of width and 7.96 mm of thickness). The moisture content of the cocoa beans was in the range between 5.64 and 6.99 (% wb) and the ash content in the range between 3.67% and 2.47% (wb). Noticeably, the fat content ratio (the most important value of cocoa bean) found in 8 varieties (TD1, TD2, TD5, TD9, TD11, TD12, TD13, TD14) was over 50%, thus these varieties are worthy chemical component especially for exported cocoa bean and industrized cultivation (extending the cultivation producing area)

An analysis of the Lowest Total Fertility Rate in Hong Kong SAR

Total Fertility Rate (TFR) in Hong Kong has dropped significantly over the past 30 years, from 2.48 births per woman in 1976 to 0.966 in 2005, which is one of the lowest in the world. It is mainly caused by the change of marital distribution which has contributed to about 56% of the decline in the total fertility rate for the period 1976-2001. Delay of marriage and reduction in the marital fertility rate have also been shown to be two major causes for the low TFR. A new measure, called a weighted total marital fertility rate (WTMFR), is introduced such that change of age at marriage and the fertility within marriage can be factored in explaining the decline of the fertility rate. The delay of marriage has contributed to about 52% of the reduction of WTMFR whereas the reduction of the fertility within marriage has accounted for the other 48%. Apparently, the proportion of women remaining single has been stabilized and leveled off recently. However, the preference of having smaller family size has become a norm rather than an exception. It is very unlikely to see a rebound of fertility among the Hong Kong women in the near future if there is no increase in marriages or births outside wedlock. Encouraging more births among married women so as to increase fertility is expected to have limited impact.Age at first marriage, decomposition, Hong Kong, Total fertility rate, Weighted total marital fertility rate

Emotional Eating and Diet-related Self-Efficacy, Motivation, and Norms in Adolescents

The objective of the current study was to examine the association between emotional eating and self-efficacy, motivation, and social norms for consumption of fruits and vegetables (F/V) and energy-dense, nutrient-poor (EDNP) foods and beverages, as well as interactions with body mass index-z score (BMI-z). Adolescents completed self-report measures of demographics, emotional eating, and dietary health behavior theory constructs. Emotional eating was associated with lower self-efficacy for consumption of F/V and for limiting EDNP foods/beverages; greater motivation for limiting of EDNP foods/beverages; lower social norms for consumption of F/V; and greater social norms for consumption of EDNP foods/beverages. There were no interactions with BMI-z. Evidence-based nutrition programs that leverage health behavior theories should be tailored to adolescents’ emotional eating

Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti

<p>Abstract</p> <p>Background</p> <p><it>S. meliloti </it>forms indeterminate nodules on the roots of its host plant alfalfa (<it>Medicago sativa</it>). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in <it>S. meliloti </it>and is the only enzyme in this half of the PHB cycle that remains uncharacterized.</p> <p>Results</p> <p>The <it>S. meliloti phaZ </it>gene was identified by <it>in silico </it>analysis, the ORF was cloned, and a <it>S. meliloti phaZ </it>mutant was constructed. This mutant exhibited increased PHB accumulation during free-living growth, even when grown under non-PHB-inducing conditions. The <it>phaZ </it>mutant demonstrated no reduction in symbiotic capacity; interestingly, analysis of the bacteroids showed that this mutant also accumulated PHB during symbiosis. This mutant also exhibited a decreased capacity to tolerate long-term carbon starvation, comparable to that of other PHB cycle mutants. In contrast to other PHB cycle mutants, the <it>S. meliloti phaZ </it>mutant did not exhibit any decrease in rhizosphere competitiveness; however, this mutant did exhibit a significant increase in succinoglycan biosynthesis.</p> <p>Conclusions</p> <p><it>S. meliloti </it>bacteroids retain the capacity to synthesize PHB during symbiosis; interestingly, accumulation does not occur at the expense of symbiotic performance. <it>phaZ </it>mutants are not compromised in their capacity to compete for nodulation in the rhizosphere, perhaps due to increased succinoglycan production resulting from upregulation of the succinoglycan biosynthetic pathway. The reduced survival capacity of free-living cells unable to access their accumulated stores of PHB suggests that PHB is a crucial metabolite under adverse conditions.</p

Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients

Assessment of genetic diversity among the main fourteen cocoa varieties in Vietnam using plant c/d sequences

This work focused on clarifying the genetic diversity of fourteen cocoa clones in Vietnam. Primers plant c and plant d were used to analyze the genetic relationship. All detected band were encoded into binary matrix and the dendrogram was generated using the program NTSYS-PC 2.1 The similarity matrix was subjected to cluster analysis by unweighted pair group method for arithmetic mean (UPGMA). The genetic clustering of 14 Cocoa varieties showed genetic differences ranged from 0 to 41%. At around 59% similarity, fourteen TD clones could be divided into three groups: A, B, and C. Group A had ten clones (TD1, TD2, TD3, TD5, TD6, TD7, TD8, TD9, TD11, TD12), in group B there were 2 clones (TD10 and TD13), and two clones belonged to group C (TD14 và TD15). Group B with 80% homologous had a relationship closer than groups A and C. Two clones in group A correlated approximately 76.4% similarity and group C showed 73% similarity rate. This result contributed important signification for Vietnamese Cocoa breeding and selection cultivar activities

Felicidad en mujeres puérperas: estudio multicéntrico en Lima Metropolitana y Callao

Objetivo: Evaluar la felicidad y los factores asociados en mujeres puérperas de Lima Metropolitana y Callao, Perú. Métodos: Estudio multicéntrico, analítico, y de corte transversal realizado en los cinco hospitales más representativos de atención pública del Ministerio de Salud en Lima Metropolitana y Callao: Instituto Nacional Materno Perinatal, Hospital Nacional Docente Madre Niño San Bartolomé, Hospital Nacional Arzobispo Loayza, Hospital Nacional 2 de Mayo y Hospital Nacional Daniel Alcides Carrión. La información fue obtenida mediante revisión de historias clínicas, cuestionarios de auto reporte y entrevista personal. La investigación fue aprobada por los Comités de Investigación y Ética de los hospitales en cuestión. Resultados: Se tomó una muestra total de 772 puérperas. La edad promedio fue de 26.7±5.9 años, 63.2% era conviviente, 79.8% estaba desempleada, 64.4% contaba con estudios secundarios completos y 62.6% refirió embarazos no planificados. La media de felicidad registrada entre las puérperas fue 31.7±5.5 puntos (rango: 11 a 40). El alfa de Cronbach fue de 0.86. En el análisis de regresión múltiple, las siguientes variables permanecieron significativas e independientemente asociadas con la felicidad: edad, estado laboral, tipo de parto, planificación del embarazo, apoyo de la pareja y amigos. Conclusiones: La felicidad es un fenómeno multifactorial. Los índices de felicidad encontrados fueron altos.El periodo postparto podría ser una buena oportunidad para explorar los sentimientos de la madre y poder implementar políticas de ayuda que puedan optimizar la salud maternoinfantil