Search results for: whole genome sequencing analysis

Authors: Parag Katira, Frederika Rentzeperis, Zuzanna Nowicka, Giada Fiandaca, Thomas Veith, Jack Farinhas, Noemi Andor

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Resource limitations shape the outcome of competitions between genetically heterogeneous pre-malignant cells. One example of such heterogeneity is in the ploidy (DNA content) of pre-malignant cells. A whole-genome duplication (WGD) transforms a diploid cell into a tetraploid one and has been detected in 28-56% of human cancers. If a tetraploid subclone expands, it consistently does so early in tumor evolution, when cell density is still low, and competition for nutrients is comparatively weak – an observation confirmed for several tumor types. WGD+ cells need more resources to synthesize increasing amounts of DNA, RNA, and proteins. To quantify resource limitations and how they relate to ploidy, we performed a PAN cancer analysis of WGD, PET/CT, and MRI scans. Segmentation of >20 different organs from >900 PET/CT scans were performed with MOOSE. We observed a strong correlation between organ-wide population-average estimates of Oxygen and the average ploidy of cancers growing in the respective organ (Pearson R = 0.66; P= 0.001). In-vitro experiments using near-diploid and near-tetraploid lineages derived from a breast cancer cell line supported the hypothesis that DNA content influences Glucose- and Oxygen-dependent proliferation-, death- and migration rates. To model how subpopulations with variable DNA content compete in the resource-limited environment of the human brain, we developed a stochastic state-space model of the brain (S3MB). The model discretizes the brain into voxels, whereby the state of each voxel is defined by 8+ variables that are updated over time: stiffness, Oxygen, phosphate, glucose, vasculature, dead cells, migrating cells and proliferating cells of various DNA content, and treat conditions such as radiotherapy and chemotherapy. Well-established Fokker-Planck partial differential equations govern the distribution of resources and cells across voxels. We applied S3MB on sequencing and imaging data obtained from a primary GBM patient. We performed whole genome sequencing (WGS) of four surgical specimens collected during the 1ˢᵗ and 2ⁿᵈ surgeries of the GBM and used HATCHET to quantify its clonal composition and how it changes between the two surgeries. HATCHET identified two aneuploid subpopulations of ploidy 1.98 and 2.29, respectively. The low-ploidy clone was dominant at the time of the first surgery and became even more dominant upon recurrence. MRI images were available before and after each surgery and registered to MNI space. The S3MB domain was initiated from 4mm³ voxels of the MNI space. T1 post and T2 flair scan acquired after the 1ˢᵗ surgery informed tumor cell densities per voxel. Magnetic Resonance Elastography scans and PET/CT scans informed stiffness and Glucose access per voxel. We performed a parameter search to recapitulate the GBM’s tumor cell density and ploidy composition before the 2ⁿᵈ surgery. Results suggest that the high-ploidy subpopulation had a higher Glucose-dependent proliferation rate (0.70 vs. 0.49), but a lower Glucose-dependent death rate (0.47 vs. 1.42). These differences resulted in spatial differences in the distribution of the two subpopulations. Our results contribute to a better understanding of how genomics and microenvironments interact to shape cell fate decisions and could help pave the way to therapeutic strategies that mimic prognostically favorable environments.

Keywords: tumor evolution, intra-tumor heterogeneity, whole-genome doubling, mathematical modeling

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Authors: Hana Barak, Alex Sivan, Ariel Kushmaro

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Microorganisms are the most diverse and abundant life forms on Earth and account for a large portion of the Earth’s biomass and biodiversity. To date though, our knowledge regarding microbial life is lacking, as it is based mainly on information from cultivated organisms. Indeed, microbiologists have borrowed from astrophysics and termed the ‘uncultured microbial majority’ as ‘microbial dark matter’. The realization of how diverse and unexplored microorganisms are, actually stems from recent advances in molecular biology, and in particular from novel methods for sequencing microbial small subunit ribosomal RNA genes directly from environmental samples termed next-generation sequencing (NGS). This has led us to use NGS that generates several gigabases of sequencing data in a single experimental run, to identify and classify environmental samples of microorganisms. In metagenomics sequencing analysis (both 16S and shotgun), sequences are compared to reference databases that contain only small part of the existing microorganisms and therefore their taxonomy assignment may reveal groups of unknown microorganisms or origins. These unknowns, or the ‘microbial sequences dark matter’, are usually ignored in spite of their great importance. The goal of this work was to develop an improved bioinformatics method that enables more complete analyses of the microbial communities in numerous environments. Therefore, NGS was used to identify previously unknown microorganisms from three different environments (industrials wastewater, Negev Desert’s rocks and water wells at the Arava valley). 16S rRNA gene metagenome analysis of the microorganisms from those three environments produce about ~4 million reads for 75 samples. Between 0.1-12% of the sequences in each sample were tagged as ‘Unassigned’. Employing relatively simple methodology for resequencing of original gDNA samples through Sanger or MiSeq Illumina with specific primers, this study demonstrates that the mysterious ‘Unassigned’ group apparently contains sequences of candidate phyla. Those unknown sequences can be located on a phylogenetic tree and thus provide a better understanding of the ‘sequences dark matter’ and its role in the research of microbial communities and diversity. Studying this ‘dark matter’ will extend the existing databases and could reveal the hidden potential of the ‘microbial dark matter’.

Keywords: bacteria, bioinformatics, dark matter, Next Generation Sequencing, unknown

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Authors: Bhaven N. Tandel, Athira Rajeev

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Sequencing batch reactor process is a suspended growth process operating under non-steady state conditions which utilizes a fill and draw reactor with complete mixing during the batch reaction step (after filling) and where the subsequent steps of aeration and clarification occur in the same tank. All sequencing batch reactor systems have five steps in common, which are carried out in sequence as follows, (1) fill (2) react (3) settle (sedimentation/clarification) (4) draw (decant) and (5) idle. The study was carried out in a sequencing batch reactor of dimensions 44cmx30cmx70cm with a working volume of 40 L. Mechanical stirrer of 100 rpm was used to provide continuous mixing in the react period and oxygen was supplied by fish tank aerators. The duration of a complete cycle of sequencing batch reactor was 8 hours. The cycle period was divided into different phases in sequence as follows-0.25 hours fill phase, 6 hours react period, 1 hour settling phase, 0.5 hours decant period and 0.25 hours idle phase. The study consisted of two runs, run 1 and run 2. Run 1 consisted of 6 hours aerobic react period and run 2 consisted of 3 hours aerobic react period followed by 3 hours anoxic react period. The influent wastewater used for the study had COD, BOD, NH3-N and TKN concentrations of 308.03±48.94 mg/L, 100.36±22.05 mg/L, 14.12±1.18 mg/L, and 24.72±2.21 mg/L respectively. Run 1 had an average COD removal efficiency of 41.28%, BOD removal efficiency of 56.25%, NH3-N removal efficiency of 86.19% and TKN removal efficiency of 54.4%. Run 2 had an average COD removal efficiency of 63.19%, BOD removal efficiency of 73.85%, NH3-N removal efficiency of 90.74% and TKN removal efficiency of 65.25%. It was observed that run 2 gave better performance than run 1 in the removal of COD, BOD and TKN.

Keywords: municipal waste water, aerobic, anoxic, sequencing batch reactor

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Authors: M. Ottenbrite, G. Yilmaz, J. Devenish, M. Kang, H. Dan, M. Lin, C. Lau, C. Carrillo, K. Bessonov, J. Nash, E. Topp, J. Guan

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Consumption of food contaminated by antibiotic-resistant (AR) bacteria may lead to the transmission of AR genes in the gut microbiota and cause AR bacterial infection, a significant public health concern. However, information is limited on if and how background microbes from the food matrix (food microbes) may influence resistance transmission. Thus, we assessed the colonization of a β-lactam resistant Salmonella Heidelberg strain (donor) and a β-lactam susceptible S. Typhimurium strain (recipient) and the transfer of the resistance genes in the mouse gut in the presence or absence of food microbes that were derived from washing freshly-harvested carrots. Mice were pre-treated with streptomycin and then inoculated with both donor and recipient bacteria or recipient only. Fecal shedding of the donor, recipient, and transconjugant bacteria was enumerated using selective culture techniques. Transfer of AR genes was confirmed by whole genome sequencing. Gut microbial composition was determined by 16s rRNA amplicon sequencing. Significantly lower numbers of donors and recipients were shed from mice that were inoculated with food microbes compared to those without food microbe inoculation. S. Typhimurium transconjugants were only recovered from mice without inoculation of food microbes. A significantly higher survival rate was in mice with vs. without inoculation of food microbes. The results suggest that the food microbes may compete with both the donor and recipient Salmonella, limit their growth and reduce transmission of the β-lactam resistance gene in the mouse gut.

Keywords: antibiotic resistance, gene transfer, gut microbiota, Salmonella infection

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Authors: Shama, Asif Mahmood, Wen Zhang

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Cardiovirus is a genus of viruses belonging to the family Picornaviridae. Here, we used viral metagenomic techniques to detect the viral nucleic acid in the fecal samples from wild rats in Zhenjiang city in China. Fecal samples were collected from 20 wild rats and pooled into four sample pools and then subjected to library construction, which were then sequenced on the Illumina MiSeq platform. The sequenced reads were analyzed using a viral metagenomic analysis pipeline. A cardiovirus from the feces of a wild rat was identified, named amzj-2018, of which the complete genome was acquired. Phylogenetic analysis based on the complete amino acid sequence of polyprotein revealed that amzj-2018 formed a separate branch located between clusters of Saffold virus and Rat Theilovirus 1 (RTV-1). Phylogenetic analysis based on different regions of the polyproteins, including P1, P2, P3, and P2+P3, respectively, showed discordant trees, where the tree based on the P3 region indicated that amzj-2018 clustered separately between Theiler's murine encephalomyelitis virus and RTV-1. The complete genome of a cardiovirus was determined from the feces of wild rats, which belonged to a novel type of cardiovirus based on phylogenetic analysis. Whether it is associated with disease needs further investigation.

Keywords: cardioviruses, viral metagenomics, novel viruses, virus-host interaction

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Authors: Mohammad Ahangari

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Schizophrenia is a psychiatric disorder with symptoms that include cognitive deficits and nicotine has been suggested to have an effect on cognition. In recent years, the advents of Genome-Wide Association Studies(GWAS) has evolved our understanding about the genetic causes of complex disorders such as schizophrenia and studying the role of genome-wide significant genes could potentially lead to the development of new therapeutic agents for treatment of cognitive deficits in schizophrenia. The current study identified six Single Nucleotide Polymorphisms (SNP) from schizophrenia and smoking GWAS that are located on or in close proximity to the nicotinic receptor gene cluster (CHRN) and studied their association with cognition in an Irish sample of 1297 cases and controls using linear regression analysis. Further on, the interaction between CHRN gene cluster and Dopamine receptor D2 gene (DRD2) during working memory was investigated. The effect of these polymorphisms on nicotinic and dopaminergic neurotransmission, which is disrupted in schizophrenia, have been characterized in terms of their effects on memory, attention, social cognition and IQ as measured by a neuropsychological test battery and significant effects in two polymorphisms were found across global IQ domain of the test battery.

Keywords: cognition, dopamine, GWAS, nicotine, schizophrenia, SNPs

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Authors: Amin Mojiri, Hamidi Abdul Aziz

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Leachate is created while water penetrates through the waste in a landfill, carrying some forms of pollutants. In literature, for treatment of wastewater and leachate, different ways of biological treatment were used. Sequencing batch reactor (SBR) is a kind of biological treatment. This study investigated the co-treatment of landfill leachate and domestic waste water by SBR and powdered activated carbon augmented (PAC) SBR process. The response surface methodology (RSM) and central composite design (CCD) were employed. The independent variables were aeration rate (L/min), contact time (h), and the ratio of leachate to wastewater mixture (%; v/v)). To perform an adequate analysis of the aerobic process, three dependent parameters, i.e. COD, color, and ammonia-nitrogen (NH3-N or NH4-N) were measured as responses. The findings of the study indicated that the PAC-SBR showed a higher performance in elimination of certain pollutants, in comparison with SBR. With the optimal conditions of aeration rate (0.6 L/min), leachate to waste water ratio (20%), and contact time (10.8 h) for the PAC-SBR, the removal efficiencies for color, NH3-N, and COD were 72.8%, 98.5%, and 65.2%, respectively.

Keywords: co-treatment, landfill Leachate, wastewater, sequencing batch reactor, activate carbon

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Authors: Rabia Faridi, Rizwana Maqbool, Umara Sahar Rana, Zaheer Ahmad

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Climate change impacts agriculture, notably in Germany, where spring faba beans predominate. However, improved winter hardiness aligns with milder winters, enabling autumn-sown varieties. Genetic resistance to Ascochyta blight is vital for crop integration. Traditional breeding faces challenges due to complex inheritance. This study assessed 224 homozygous faba bean lines for Ascochyta resistance traits. To achieve h²>70%, 12 replicates were required (realized h²=87%). Genetic variation and strong trait correlations were observed. Five lines outperformed 29H, while three were highly susceptible. A genome-wide association study (GWAS) with 188 inbred lines and 2058 markers, including 17 guide SNP markers, identified 12 markers associated with resistance traits, potentially indicating new resistance genes. One guide marker (Vf-Mt1g014230-001) on chromosome III validated a known QTL. The guided marker approach complemented GWAS, facilitating marker-assisted selection for Ascochyta resistance. The Göttingen Winter Bean Population offers promise for resistance breeding.

Keywords: genome wide association studies, marker assisted breeding, faba bean, ascochyta blight

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Authors: Ruchi Pathania, Ahmad Ahmad, Shireesh Srivastava

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Cyanobacteria is a promising microbe that can capture and convert atmospheric CO₂ and light into valuable industrial bio-products like biofuels, biodegradable plastics, etc. Among their most attractive traits are faster autotrophic growth, whole year cultivation using non-arable land, high photosynthetic activity, much greater biomass and productivity and easy for genetic manipulations. Cyanobacteria store carbon in the form of glycogen which can be hydrolyzed to release glucose and fermented to form bioethanol or other valuable products. Marine cyanobacterial species are especially attractive for countries with scarcity of freshwater. We recently identified a marine native cyanobacterium Synechococcus sp. BDU 130192 which has good growth rate and high level of polyglucans accumulation compared to Synechococcus PCC 7002. In this study, firstly we sequenced the whole genome and the sequences were annotated using the RAST server. Genome scale metabolic model (GSMM) was reconstructed through COBRA toolbox. GSMM is a computational representation of the metabolic reactions and metabolites of the target strain. GSMMs construction through the application of Flux Balance Analysis (FBA), which uses external nutrient uptake rates and estimate steady state intracellular and extracellular reaction fluxes, including maximization of cell growth. The model, which we have named isyn942, includes 942 reactions and 913 metabolites having 831 metabolic, 78 transport and 33 exchange reactions. The phylogenetic tree obtained by BLAST search revealed that the strain was a close relative of Synechococcus PCC 7002. The flux balance analysis (FBA) was applied on the model iSyn942 to predict the theoretical yields (mol product produced/mol CO₂ consumed) for native and non-native products like acetone, butanol, etc. under phototrophic condition by applying metabolic engineering strategies. The reported strain can be a viable strain for biotechnological applications, and the model will be helpful to researchers interested in understanding the metabolism as well as to design metabolic engineering strategies for enhanced production of various bioproducts.

Keywords: cyanobacteria, flux balance analysis, genome scale metabolic model, metabolic engineering

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Authors: Priyanka Dargode, Suhas Gore, Manju Sharma, Arvind Lali

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Anaerobic digestion has been widely used for production of methane from different biowastes. However, the complexity of microbial communities involved in the process is poorly understood. The performance of biogas production process concerning the process productivity is closely coupled to its microbial community structure and syntrophic interactions amongst the community members. The present study aims at understanding taxonomic variations occurring in any starter inoculum when acclimatised to different lignocellulosic biomass (LBM) feedstocks relating to time of digestion. The work underlines use of high throughput Next Generation Sequencing (NGS) for validating the changes in taxonomic patterns of microbial communities. Biomethane Potential (BMP) batches were set up with different pretreated and non-pretreated LBM residues using the same microbial consortium and samples were withdrawn for studying the changes in microbial community in terms of its structure and predominance with respect to changes in metabolic profile of the process. DNA of samples withdrawn at different time intervals with reference to performance changes of the digestion process, was extracted followed by its 16S rRNA amplicon sequencing analysis using Illumina Platform. Biomethane potential and substrate consumption was monitored using Gas Chromatography(GC) and reduction in COD (Chemical Oxygen Demand) respectively. Taxonomic analysis by QIIME server data revealed that microbial community structure changes with different substrates as well as at different time intervals. It was observed that biomethane potential of each substrate was relatively similar but, the time required for substrate utilization and its conversion to biomethane was different for different substrates. This could be attributed to the nature of substrate and consequently the discrepancy between the dominance of microbial communities with regards to different substrate and at different phases of anaerobic digestion process. Knowledge of microbial communities involved would allow a rational substrate specific consortium design which will help to reduce consortium adaptation period and enhance the substrate utilisation resulting in improved efficacy of biogas process.

Keywords: amplicon sequencing, biomethane potential, community predominance, taxonomic analysis

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Authors: Abul B. M. M. K. Islam, Nuria Lopez-Bigas, Elizaveta V. Benevolenskaya

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RBP2 has shown to be important for cell differentiation control through epigenetic mechanism. The main aim of the present study is genome-wide location analysis of human RBP2 isoforms that differ in a histone-binding domain by ChIPseq. It is conceivable that the larger isoform (LI) of RBP2, which contains a specific H3K4me3 interacting domain, differs from the smaller isoform (SI) in genomic location, may account for the observed diversity in RBP2 function. To distinguish the two RBP2 isoforms, we used the fact that the SI lacks the C-terminal PHD domain and hence used the antibodies detecting both RBP2 isoforms (AI) through a common central domain, and the antibodies detecting only LI but not SI, through a C-terminal PHD domain. Overall our analysis suggests that RBP2 occupies about 77 nucleotides and binds GC rich motifs of active genes, does not bind to centromere, telomere, or enhancer regions, and binding sites are conserved compare to random. A striking difference between the only-SI and only-LI is that a large number of only-SI peaks are located in CpG islands and close to TSS compared to only-LI peaks. Enrichment analysis of the related genes indicates that several oncogenic pathways and metabolic pathways/processes are significantly enriched among only-SI/AI targets, but not LI/only-LI peak’s targets.

Keywords: bioinformatics, cancer, ChIP-seq, KDM5A

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Authors: Abdul Musaweer Habib, Habibul Hasan Mazumder, Saiful Islam, Sohel Sikder, Omar Faruk Sikder

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The plethora of genome sequence information of bacteria in recent times has ushered in many novel strategies for antibacterial drug discovery and facilitated medical science to take up the challenge of the increasing resistance of pathogenic bacteria to current antibiotics. In this study, we adopted subtractive genomics approach to analyze the whole genome sequence of the Fusobacterium nucleatum, a human oral pathogen having association with colorectal cancer. Our study divulged 1499 proteins of Fusobacterium nucleatum, which has no homolog in human genome. These proteins were subjected to screening further by using the Database of Essential Genes (DEG) that resulted in the identification of 32 vitally important proteins for the bacterium. Subsequent analysis of the identified pivotal proteins, using the KEGG Automated Annotation Server (KAAS) resulted in sorting 3 key enzymes of F. nucleatum that may be good candidates as potential drug targets, since they are unique for the bacterium and absent in humans. In addition, we have demonstrated the 3-D structure of these three proteins. Finally, determination of ligand binding sites of the key proteins as well as screening for functional inhibitors that best fitted with the ligands sites were conducted to discover effective novel therapeutic compounds against Fusobacterium nucleatum.

Keywords: colorectal cancer, drug target, Fusobacterium nucleatum, homology modeling, ligands

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Authors: Naoki Yamamoto, Hidenobu Ozaki, Taiichiro Ookawa, Youming Liu, Kazunori Okada, Aiping Zheng

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Rice sheath blight is one of the destructive fungal diseases in rice. We have thought that rice sheath blight resistance is a polygenic trait. Host-pathogen interactions and secondary metabolites such as lignin and phytoalexins are likely to be involved in defense against R. solani. However, to our knowledge, it is still unknown how sheath blight resistance can be enhanced in rice breeding. To seek for an alternative genetic factor that contribute to sheath blight resistance, we mined relevant allelic variations from rice core collections created in Japan. Based on disease lesion length on detached leaf sheath, we selected 30 varieties of the top tail-end and the bottom tail-end, respectively, from the core collections to perform genome-wide association mapping. Re-sequencing reads for these varieties were used for calling single nucleotide polymorphisms among the 60 varieties to create a SNP panel, which contained 1,137,131 homozygous variant sites after filitering. Association mapping highlighted a locus on the long arm of chromosome 11, which is co-localized with three sheath blight QTLs, qShB11-2-TX, qShB11, and qSBR-11-2. Based on the localization of the trait-associated alleles, we identified an ankyryn repeat-containing protein gene (ANK-M) as an uncharacterized candidate factor for rice sheath blight resistance. Allelic distributions for ANK-M in the whole rice population supported the reliability of trait-allele associations. Gene expression characteristics were checked to evaluiate the functionality of ANK-M. Since an ANK-M homolog (OsPIANK1) in rice seems a basal defense regulator against rice blast and bacterial leaf blight, ANK-M may also play a role in the rice immune system.

Keywords: allele mining, GWAS, QTL, rice sheath blight

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Authors: Zohar Manber, Ran Elkon

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Accumulation of somatic mutations (SMs) in the genome is a major driving force of cancer development. Most SMs in the tumor's genome are functionally neutral; however, some cause damage to critical processes and provide the tumor with a selective growth advantage (termed cancer driver mutations). Current research on functional significance of SMs is mainly focused on finding alterations in protein coding sequences. However, the exome comprises only 3% of the human genome, and thus, SMs in the noncoding genome significantly outnumber those that map to protein-coding regions. Although our understanding of noncoding driver SMs is very rudimentary, it is likely that disruption of regulatory elements in the genome is an important, yet largely underexplored mechanism by which somatic mutations contribute to cancer development. The expression of most human genes is controlled by multiple enhancers, and therefore, it is conceivable that regulatory SMs are distributed across different enhancers of the same target gene. Yet, to date, most statistical searches for regulatory SMs have considered each regulatory element individually, which may reduce statistical power. The first challenge in considering the cumulative activity of all the enhancers of a gene as a single unit is to map enhancers to their target promoters. Such mapping defines for each gene its set of regulating enhancers (termed "set of regulatory elements" (SRE)). Considering multiple enhancers of each gene as one unit holds great promise for enhancing the identification of driver regulatory SMs. However, the success of this approach is greatly dependent on the availability of comprehensive and accurate enhancer-promoter (E-P) maps. To date, the discovery of driver regulatory SMs has been hindered by insufficient sample sizes and statistical analyses that often considered each regulatory element separately. In this study, we analyzed more than 2,500 whole-genome sequence (WGS) samples provided by The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC) in order to identify such driver regulatory SMs. Our analyses took into account the combinatorial aspect of gene regulation by considering all the enhancers that control the same target gene as one unit, based on E-P maps from three genomics resources. The identification of candidate driver noncoding SMs is based on their recurrence. We searched for SREs of genes that are "hotspots" for SMs (that is, they accumulate SMs at a significantly elevated rate). To test the statistical significance of recurrence of SMs within a gene's SRE, we used both global and local background mutation rates. Using this approach, we detected - in seven different cancer types - numerous "hotspots" for SMs. To support the functional significance of these recurrent noncoding SMs, we further examined their association with the expression level of their target gene (using gene expression data provided by the ICGC and TCGA for samples that were also analyzed by WGS).

Keywords: cancer genomics, enhancers, noncoding genome, regulatory elements

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Authors: Jae-Hyung Jin, Kyung-Tae Lee, Yee-Seul So, Eunji Jeong, Yeonseon Lee, Dongpil Lee, Dong-Gi Lee, Yong-Sun Bahn

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Cryptococcus neoformans causes cryptococcal meningoencephalitis mainly in immunocompromised patients as well as immunocompetent people. But therapeutic options are limited to treat cryptococcosis. Some signaling pathways including cyclic AMP pathway, MAPK pathway, and calcineurin pathway play a central role in the regulation of the growth, differentiation, and virulence of C. neoformans. To understand signaling networks regulating the virulence of C. neoformans, we selected the 114 putative phosphatase genes, one of the major components of signaling networks, in the genome of C. neoformans. We identified putative phosphatases based on annotation in C. neoformans var. grubii genome database provided by the Broad Institute and National Center for Biotechnology Information (NCBI) and performed a BLAST search of phosphatases of Saccharomyces cerevisiae, Aspergillus nidulans, Candida albicans and Fusarium graminearum to Cryptococcus neoformans. We classified putative phosphatases into 14 groups based on InterPro phosphatase domain annotation. Here, we constructed 170 signature-tagged gene-deletion strains through homologous recombination methods for 91 putative phosphatases. We examined their phenotypic traits under 30 different in vitro conditions, including growth, differentiation, stress response, antifungal resistance and virulence-factor production.

Keywords: human fungal pathogen, phosphatase, deletion library, functional genomics

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Authors: Rafael Estrada, Cristian Ruiz Rueda

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Carbapenems are last-resort antibiotics used in clinical settings to treat antibiotic-resistant bacterial infections. Thus, the emergence and spread of resistance to carbapenems is a major public health concern. Here, we have studied a carbapenem-resistant Aeromonas veronii strain previously isolated from a water sample from Sam Simeon Creek (Hearst San Simeon State Park, CA). Analysis of this isolate using disk-diffusion, CarbaNP, eCIM and mCIM assays revealed that it was resistant to amoxicillin-clavulanic acid and all carbapenems tested and that this isolate produced a potentially novel carbapenemase of the Metallo-β-lactamase family. Whole genome sequencing analysis revealed that this A. veronii isolate carries a novel variant of the blacₚₕₐ class β-carbapenemase gene that was closely related to the blacₚₕₐ₇ gene of Aeromonas jandaei. This isolate also carried a novel variant of the blaₒₓₐ class D carbapenemase gene that was most closely related to the blaₒₓₐ-₉₁₂ gene found in other Aeromonas veronii isolates. Finally, we also identified a novel class C β-lactamase gene moderately related to the blaFₒₓ-₁₇ gene of Providencia stuartii and other blaFₒₓ variants identified in Klebsiella pneumoniae, Escherichia coli and other Enterobacteriaceae. Overall, our findings reveal that environmental isolates are an important reservoir of multiple carbapenemases and other β-lactamases of clinical significance.

Keywords: β-lactamases, carbapenem, antibiotic-resistant, aeromonas veronii

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Authors: Alyaa Abdlwahab

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Cutaneous leishmaniasis represents a serious health problem in Syria; this problem has become noticeably aggravated after the civil war in the country. Leishmania tropica parasite is the main cause of cutaneous leishmaniasis in Syria. In order to control the disease, we need an effective vaccine against leishmania parasite. DNA vaccination remains one of the favorable approaches that have been used to face cutaneous leishmaniasis. Ribosomal protein S4 is responsible for important roles in Leishmania parasite life. DNA vaccine based on S4 gene has been used against infections by many species of Leishmania parasite but leishmania tropica parasite, so this gene represents a good candidate for DNA vaccine construction. After proving the existence of ribosomal protein S4 gene in a Syrian strain of Leishmania tropica (LCED Syrian 01), sequencing it and cloning it into pCI plasmid, BALB/C mice were inoculated with pCI-S4 DNA vaccine. The immune response was determined by monitoring the lesion progression in inoculated BALB/C mice for six weeks after challenging mice with Leishmania tropica (LCED Syrian 01) parasites. IL-12, IFN-γ, and IL-4 were quantified in draining lymph nodes (DLNa) of the immunized BALB/C mice by using the RT-qPCR technique. The parasite burden was calculated in the final week for the footpad lesion and the DLNs of the mice. This study proved the existence and the expression of the ribosomal protein S4 gene in Leishmania tropica (LCED Syrian 01) promastigotes. The sequence of ribosomal protein cDNA S4 gene was determined and published in Genbank; the gene size was 822 bp. Expression was also demonstrated at the level of cDNA. Also, this study revealed that pCI-S4 DNA vaccine induces TH1\TH2 response in immunized mice; this response prevents partially developing a dermal lesion of Leishmania.

Keywords: ribosomal protein S4, DNA vaccine, Leishmania tropica, BALB\c

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Authors: Satwik Majumder, Dongyun Jung, Jennifer Ronholm, Saji George

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Bovine mastitis is the most common infectious disease in dairy cattle, with major economic implications for the dairy industry worldwide. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from dairy farms is vital not only for animal husbandry but also for public health. In this study, the prevalence of AMR in 113 Escherichia coli isolates from cases of bovine clinical mastitis in Canada was investigated. Kirby-Bauer disk diffusion test with 18 antibiotics and microdilution method with three heavy metals (copper, zinc, and silver) was performed to determine the antibiotic and heavy-metal susceptibility. Resistant strains were assessed for efflux and ß-lactamase activities besides assessing biofilm formation and hemolysis. Whole-genome sequences for each of the isolates were examined to detect the presence of genes corresponding to the observed AMR and virulence factors. Phenotypic analysis revealed that 32 isolates were resistant to one or more antibiotics, and 107 showed resistance against at least one heavy metal. Quinolones and silver were the most efficient against the tested isolates. Among the AMR isolates, AcrAB-TolC efflux activity and ß-lactamase enzyme activities were detected in 13 and 14 isolates, respectively. All isolates produced biofilm but with different capacities, and 33 isolates showed α-hemolysin activity. A positive correlation (Pearson r = +0.89) between efflux pump activity and quantity of biofilm was observed. Genes associated with aggregation, adhesion, cyclic di-GMP, quorum sensing were detected in the AMR isolates, corroborating phenotype observations. This investigation showed the prevalence of AMR in E. coli isolates from bovine clinical mastitis. The results also suggest the inadequacy of antimicrobials with a single mode of action to curtail AMR bacteria with multiple mechanisms of resistance and virulence factors. Therefore, it calls for combinatorial therapy for the effective management of AMR infections in dairy farms and combats its potential transmission to the food supply chain through milk and dairy products.

Keywords: antimicrobial resistance, E. coli, bovine mastitis, antibiotics, heavy-metals, efflux pump, ß-lactamase enzyme, biofilm, whole-genome sequencing

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Authors: M. L. Mangena, N. Mokoena, K. Rashamuse, M. G. Tlou

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Tertiary alcohol ester (TAE) hydrolases are a group of esterases (EC 3.1.1.-) that catalyze the kinetic resolution of TAEs and as a result, they are sought-after for the production of optically pure tertiary alcohols (TAs) which are useful as building blocks for number biologically active compounds. What sets these enzymes apart is, the presence of a GGG(A)X-motif in the active site which appears to be the main reason behind their activity towards the sterically demanding TAEs. The genome of Pseudomonas syringae pv. maculicola (Psm) comprises a multitude of genes that encode esterases. We therefore, hypothesize that some of these genes encode TAE hydrolases. In this study, Psm was screened for TAE hydrolase activity using the linalyl acetate (LA) plate assay and a positive reaction was observed. As a result, the genome of Psm was screened for esterases with a GGG(A)X-motif using the motif search tool and two potential TAE hydrolase genes (PsmEST1 and 2, 1100 and 1000bp, respectively) were identified, PsmEST1 was amplified by PCR and the gene sequenced for confirmation. Analysis of the sequence data with the SingnalP 4.1 server revealed that the protein comprises a signal peptide (22 amino acid residues) on the N-terminus. Primers specific for the gene encoding the mature protein (without the signal peptide) were designed such that they contain NdeI and XhoI restriction sites for directional cloning of the PCR products into pET28a. The gene was expressed in E. coli JM109 (DE3) and the clones screened for TAE hydrolase activity using the LA plate assay. A positive clone was selected, overexpressed and the protein purified using nickel affinity chromatography. The activity of the esterase towards LA was confirmed using thin layer chromatography.

Keywords: hydrolases, tertiary alcohol esters, tertiary alcohols, screening, Pseudomonas syringae pv., maculicola genome, esterase activity, linalyl acetate

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Authors: Parvaneh Mahmoudi, Ahmad Moeni, Seyed Mojtaba Khayam Nekoei, Mohsen Mardi, Mehrshad Zeinolabedini, Ghasem Hosseini Salekdeh

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Saffron (Crocus sativus L.) is one of the most important spice and medicinal plants. The three-branch style of C. sativus flowers are the most important economic part of the plant and known as saffron, which has several medicinal properties. Despite the economic and biological significance of this plant, knowledge about its molecular characteristics is very limited. In the present study, we, for the first time, constructed a comprehensive dataset for C. sativus stigma through de novo transcriptome sequencing. We performed de novo transcriptome sequencing of C. sativus stigma using the Illumina paired-end sequencing technology. A total of 52075128 reads were generated and assembled into 118075 unigenes, with an average length of 629 bp and an N50 of 951 bp. A total of 66171unigenes were identified, among them, 66171 (56%) were annotated in the non-redundant National Center for Biotechnology Information (NCBI) database, 30938 (26%) were annotated in the Swiss-Prot database, 10273 (8.7%) unigenes were mapped to 141 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, while 52560 (44%) and 40756 (34%) unigenes were assigned to Gen Ontology (GO) categories and Eukaryotic Orthologous Groups of proteins (KOG), respectively. In addition, 65 candidate genes involved in three stages of crocin biosynthesis were identified. Finally, transcriptome sequencing of saffron stigma was used to identify 6779 potential microsatellites (SSRs) molecular markers. High-throughput de novo transcriptome sequencing provided a valuable resource of transcript sequences of C. sativus in public databases. In addition, most of candidate genes potentially involved in crocin biosynthesis were identified which could be further utilized in functional genomics studies. Furthermore, numerous obtained SSRs might contribute to address open questions about the origin of this amphiploid spices with probable little genetic diversity.

Keywords: saffron, transcriptome, NGS, bioinformatic

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Authors: Atef Ibrahim, Hamed Elsimary, Abdullah Aljumah, Fayez Gebali

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This paper presents a novel semi-systolic array architecture for an optimized parallel sequence alignment algorithm. This architecture has the advantage that it can be modified to be reused for multiple pass processing in order to increase the number of processing elements that can be packed into a single FPGA and to increase the number of sequences that can be aligned in parallel in a single FPGA. This resolves the potential problem of many FPGA resources left unused for designs that have large values of short read length. When using the previously published conventional hardware design. FPGA implementation results show that, for large values of short read lengths (M>128), the proposed design has a slightly higher speed up and FPGA utilization over the the conventional one.

Keywords: bioinformatics, genome sequence alignment, re-sequencing applications, systolic array

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Authors: Martynova Alina, Lyubov Buzoleva

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Streptococcus pneumoniae is the causative agent of pneumonias and meningitids throught all the world. Having high genetic diversity, this microorganism can cause different clinical forms of pneumococcal infections and microbiologically it is really difficult diagnosed by routine methods. Also, epidemiological surveillance requires more developed methods of molecular typing because the recent method of serotyping doesn't allow to distinguish invasive and non-invasive isolates properly. Non-coding genome of bacteria seems to be the interesting source for seeking of highly distinguishable markers to discriminate the subspecies of such a variable bacteria as Streptococcus pneumoniae. Technically, we proposed scheme of discrimination of S.pneumoniae strains with amplification of non-coding region (SP_1932) with the following restriction with 2 types of enzymes of Alu1 and Mn1. Aim: This research aimed to compare different methods of typing and their application for molecular epidemiology purposes. Methods: we analyzed population of 100 strains of S.pneumoniae isolated from different patients by different molecular epidemiology methods such as pulse-field gel electophoresis (PFGE), restriction polymorphism analysis (RFLP) and multilolocus sequence typing (MLST), and all of them were compared with classic typing method as serotyping. The discriminative power was estimated with Simpson Index (SI). Results: We revealed that the most discriminative typing method is RFLP (SI=0,97, there were distinguished 42 genotypes).PFGE was slightly less discriminative (SI=0,95, we identified 35 genotypes). MLST is still the best reference method (SI=1.0). Classic method of serotyping showed quite weak discriminative power (SI=0,93, 24 genotypes). In addition, sensivity of RFLP was 100%, specificity was 97,09%. Conclusion: the most appropriate method for routine epidemiology surveillance is RFLP with non-coding region of Streptococcsu pneumoniae, then PFGE, though in some cases these results should be obligatory confirmed by MLST.

Keywords: molecular epidemiology typing, non-coding genome, Streptococcus pneumoniae, MLST

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Authors: Piotr J. Balwierz, Mikhail Pachkov, Phil Arnold, Andreas J. Gruber, Mihaela Zavolan, Erik van Nimwegen

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Understanding the key players and interactions in the regulatory networks that control gene expression and chromatin state across different cell types and tissues in metazoans remains one of the central challenges in systems biology. Our laboratory has pioneered a number of methods for automatically inferring core gene regulatory networks directly from high-throughput data by modeling gene expression (RNA-seq) and chromatin state (ChIP-seq) measurements in terms of genome-wide computational predictions of regulatory sites for hundreds of transcription factors and micro-RNAs. These methods have now been completely automated in an integrated webserver called ISMARA that allows researchers to analyze their own data by simply uploading RNA-seq or ChIP-seq data sets and provides results in an integrated web interface as well as in downloadable flat form. For any data set, ISMARA infers the key regulators in the system, their activities across the input samples, the genes and pathways they target, and the core interactions between the regulators. We believe that by empowering experimental researchers to apply cutting-edge computational systems biology tools to their data in a completely automated manner, ISMARA can play an important role in developing our understanding of regulatory networks across metazoans.

Keywords: gene expression analysis, high-throughput sequencing analysis, transcription factor activity, transcription regulation

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Authors: Amin Mojiri, Akiyoshi Ohashi, Tomonori Kindaichi

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Synthetic domestic wastewater was treated via combining treatment methods, including electrochemical oxidation, adsorption, and sequencing batch reactor (SBR). In the upper part of the reactor, an anode and a cathode (Ti/RuO₂-IrO₂) were organized in parallel for the electrochemical oxidation procedure. Sodium sulfate (Na₂SO₄) with a concentration of 2.5 g/L was applied as the electrolyte. The voltage and current were fixed on 7.50 V and 0.40 A, respectively. Then, 15% working value of the reactor was filled by activated sludge, and 85% working value of the reactor was added with synthetic wastewater. Powdered cockleshell, 1.5 g/L, was added in the reactor to do ion-exchange. Response surface methodology was employed for statistical analysis. Reaction time (h) and pH were considered as independent factors. A total of 97.0% biochemical oxygen demand, 99.9% phosphorous and 88.6% cadmium were eliminated at the optimum reaction time (80.0 min) and pH (6.4).

Keywords: adsorption, electrochemical oxidation, metals, SBR

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Authors: Manasvi Pinnaka, Brianna Chrisman

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Cystic fibrosis patients often develop lung infections that range anywhere in severity from mild to life-threatening due to the presence of thick and sticky mucus that fills their airways. Since many of these infections are chronic, they not only affect a patient’s ability to breathe but also increase the chances of mortality by respiratory failure. With a publicly available dataset of DNA sequences from bacterial species in the lung microbiome of cystic fibrosis patients, the correlations between different microbial species in the lung and the extent of deterioration of lung function were investigated. 16S sequencing technologies were used to determine the microbiome composition of the samples in the dataset. For the statistical analyses, referencing helped distinguish between taxonomies, and the proportions of certain taxa relative to another were determined. It was found that the Fusobacterium, Actinomyces, and Leptotrichia microbial types all had a positive correlation with the FEV1 score, indicating the potential displacement of these species by pathogens as the disease progresses. However, the dominant pathogens themselves, including Pseudomonas aeruginosa and Staphylococcus aureus, did not have statistically significant negative correlations with the FEV1 score as described by past literature. Examining the lung microbiology of cystic fibrosis patients can help with the prediction of the current condition of lung function, with the potential to guide doctors when designing personalized treatment plans for patients.

Keywords: bacterial infections, cystic fibrosis, lung microbiome, 16S sequencing

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Authors: Narin Salehiyan, Ramin Ghasemi Shayan

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In order to investigate coronavirus RNA replication, transcription, recombination, protein processing and transport, virion assembly, the identification of coronavirus-specific cell receptors, and polymerase processing, the manipulation of coronavirus clones and complementary DNAs (cDNAs) of defective-interfering (DI) RNAs is the subject of this chapter. The idea of the Covid genome is nonsegmented, single-abandoned, and positive-sense RNA. When compared to other RNA viruses, its size is significantly greater, ranging from 27 to 32 kb. The quality encoding the enormous surface glycoprotein depends on 4.4 kb, encoding a forcing trimeric, profoundly glycosylated protein. This takes off exactly 20 nm over the virion envelope, giving the infection the appearance-with a little creative mind of a crown or coronet. Covid research has added to the comprehension of numerous parts of atomic science as a general rule, like the component of RNA union, translational control, and protein transport and handling. It stays a fortune equipped for creating startling experiences.

Keywords: covid-19, corona, virus, genome, genetic

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Authors: Farahnaz Sadat Golestan Hashemi, Mohd Razi Ismail, Mohd Y. Rafii

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Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system can facilitate targeted genome editing in organisms. Dual or single guide RNA (gRNA) can program the Cas9 nuclease to cut target DNA in particular areas; thus, introducing concise mutations either via error-prone non-homologous end-joining repairing or via incorporating foreign DNAs by homologous recombination between donor DNA and target area. In spite of high demand of such promising technology, developing a well-organized procedure in order for reliable mining of potential target sites for gRNAs in large genomic data is still challenging. Hence, we aimed to perform high-throughput detection of target sites by specific PAMs for not only common Streptococcus pyogenes (SpCas9) but also for Neisseria meningitides (NmCas9) CRISPR-Cas systems. Previous research confirmed the successful application of such RNA-guided Cas9 orthologs for effective gene targeting and subsequently genome manipulation. However, Cas9 orthologs need their particular PAM sequence for DNA cleavage activity. Activity levels are based on the sequence of the protospacer and specific combinations of favorable PAM bases. Therefore, based on the specific length and sequence of PAM followed by a constant length of the target site for the two orthogonals of Cas9 protein, we created a reliable procedure to explore possible gRNA sequences. To mine CRISPR target sites, four different searching modes of sgRNA binding to target DNA strand were applied. These searching modes are as follows i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching, and iv) paired-gRNA searching. Finally, a complete list of all potential gRNAs along with their locations, strands, and PAMs sequence orientation can be provided for both SpCas9 as well as another potential Cas9 ortholog (NmCas9). The artificial design of potential gRNAs in a genome of interest can accelerate functional genomic studies. Consequently, the application of such novel genome editing tool (CRISPR/Cas technology) will enhance by presenting increased versatility and efficiency.

Keywords: CRISPR/Cas9 genome editing, gRNA mining, SpCas9, NmCas9

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Authors: Unchalee Inkampa, Tuanjai Somboonwiwat

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Painting process of automobiles and automobile parts, which is a continuous process based on EDP (Electrode position paint, EDP). Through EDP, all work pieces will be continuously sent to the painting process. Work process can be divided into 2 groups based on the running time: Painting Room 1 and Painting Room 2. This leads to continuous operation. The problem that arises is waiting for workloads onto Painting Room. The grading process EDP to Painting Room is a major problem. Therefore, this paper aim to develop production sequencing method by applying EDP to painting process. It also applied fixed rate launching for painting room and earliest due date (EDD) for EDP process and swap pairwise interchange for waiting time to a minimum of machine. The result found that the developed method could improve painting reduced waiting time, on time delivery, meeting customers wants and improved productivity of painting unit.

Keywords: sequencing, mixed model lines, painting process, electrode position paint

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Authors: Sandhra M. Pillai

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This article examines the relevance, difficulties, and potential applications of DNA analysis in the criminal justice system. A potent tool for connecting suspects to crime sites, clearing the innocent of wrongdoing, and resolving cold cases, DNA analysis has transformed forensic investigations. The scientific foundations of DNA analysis, including DNA extraction, sequencing, and statistical analysis, are covered in the article. To guarantee accurate and trustworthy findings, it also discusses the significance of quality assurance procedures, chain of custody, and DNA sample storage. DNA analysis has significantly advanced science, but it also brings up substantial moral and legal issues. To safeguard individual rights and uphold public confidence, privacy concerns, possible discrimination, and abuse of DNA information must be properly addressed. The paper also emphasises the effects of the criminal justice system on people and communities while highlighting the necessity of equity, openness, and fair access to DNA testing. The essay describes the obstacles and future directions for DNA analysis. It looks at cutting-edge technology like next-generation sequencing, which promises to make DNA analysis quicker and more affordable. To secure the appropriate and informed use of DNA evidence, it also emphasises the significance of multidisciplinary collaboration among scientists, law enforcement organisations, legal experts, and policymakers. In conclusion, DNA analysis has enormous potential for improving the course of criminal justice. We can exploit the potential of DNA technology while respecting the ideals of justice, fairness, and individual rights by navigating the ethical, legal, and societal issues and encouraging discussion and collaboration.

Keywords: DNA analysis, DNA evidence, reliability, validity, legal frame, admissibility, ethical considerations, impact, future direction, challenges

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Authors: Ghorbanali Mohammadi

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New approaches to sequencing mixed-model manufacturing systems are present. These approaches have attracted considerable attention due to their potential to deal with difficult optimization problems. This paper presents Multi-Objective Simulated Annealing Algorithms (MOSAA) approaches to the Just-In-Time (JIT) sequencing problem where workload-smoothing (WL) and the number of set-ups (St) are to be optimized simultaneously. Mixed-model assembly lines are types of production lines where varieties of product models similar in product characteristics are assembled. Moreover, this type of problem is NP-hard. Two annealing methods are proposed to solve the multi-objective problem and find an efficient frontier of all design configurations. The performances of the two methods are tested on several problems from the literature. Experimentation demonstrates the relative desirable performance of the presented methodology.

Keywords: scheduling, just-in-time, mixed-model assembly line, sequencing, simulated annealing

Procedia PDF Downloads 87