Search results for: Illumina DNA sequencing

Authors: Christina Killian, Katie Wall, Séamus Fanning, Guerrino Macori

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Deep-level metagenomics offers a useful technical approach to explore the three dynamic One Health axes: healthcare, domiciliary and veterinary. There is currently limited understanding of the composition of complex biofilms, natural abundance of AMR genes and gene transfer occurrence in these ecological niches. By using a newly established small-scale complex biofilm model, COMBAT has the potential to provide new information on microbial diversity, antimicrobial resistance (AMR)-encoding gene abundance, and their transfer in complex biofilms of importance to these three One Health axes. Shotgun metagenomics has been used to sample the genomes of all microbes comprising the complex communities found in each biofilm source. A comparative analysis between untreated and biocide-treated biofilms is described. The basic steps include the purification of genomic DNA, followed by library preparation, sequencing, and finally, data analysis. The use of long-read sequencing facilitates the completion of metagenome-assembled genomes (MAG). Samples were sequenced using a PromethION platform, and following quality checks, binning methods, and bespoke bioinformatics pipelines, we describe the recovery of individual MAGs to identify mobile gene elements (MGE) and the corresponding AMR genotypes that map to these structures. High-throughput sequencing strategies have been deployed to characterize these communities. Accurately defining the profiles of these niches is an essential step towards elucidating the impact of the microbiota on each niche biofilm environment and their evolution.

Keywords: COMBAT, biofilm, metagenomics, high-throughput sequencing

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Authors: Pouya Karimi, Ramin Gasemi Shayan, Parsa Sheykhzade

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Ease shotgun DNA sequencing is changing the microbial sciences. Sequencing instruments are compelling to the point that example planning is currently the key constraining element. Here, we present a microfluidic test readiness stage that incorporates the key strides in cells to grouping library test groundwork for up to 96 examples and decreases DNA input prerequisites 100-overlay while keeping up or improving information quality. The universally useful microarchitecture we show bolsters work processes with subjective quantities of response and tidy up or catch steps. By decreasing the example amount necessities, we empowered low-input (∼10,000 cells) entire genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil miniaturized scale settlements with prevalent outcomes. We additionally utilized the upgraded throughput to succession ∼400 clinical Pseudomonas aeruginosa libraries and exhibit magnificent single-nucleotide polymorphism discovery execution that clarified phenotypically watched anti-toxin opposition. Completely coordinated lab-on-chip test arrangement beats specialized boundaries to empower more extensive organization of genomics across numerous fundamental research and translational applications.

Keywords: clinical microbiology, DNA, microbiology, microbial genomics

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Authors: Habib Onsori, Somayeh Akrami, Mohammad Rahmati

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Deafness is the most common sensory disorder with the frequency of 1/1000 in many populations. Mutations in the GJB2 (CX26) gene at the DFNB1 locus on chromosome 13q12 are associated with congenital hearing loss. Approximately 80% of congenital hearing loss cases are recessively inherited and 15% dominantly inherited. Mutations of the GJB2 gene, encoding gap junction protein Connexin 26 (Cx26), are the most common cause of hereditary congenital hearing loss in many countries. This report presents two cases of different mutations from Iranian patients with bilateral hearing loss. DNA studies were performed for the GJB2 gene by PCR and sequencing methods. In one of them, direct sequencing of the gene showed a heterozygous T→C transition at nucleotide 604 resulting in a cysteine to arginine amino acid substitution at codon 202 (C202R) in the fourth extracellular domain (TM4) of the protein. The analyses indicate that the C202R mutation appeared de novo in the proband with a possible dominant effect (GenBank: KF 638275). In the other one, DNA sequencing revealed a compound heterozygous mutation (35delG, 363delC) in the Cx26 gene that is strongly associated with congenital non-syndromic hearing loss (NSHL). So screening the mutations for hearing loss individuals referring to genetics counseling centers before marriage and or pregnancy is recommended.

Keywords: CX26, deafness, GJB2, mutation

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Authors: Xiaoxing Ye

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To investigate complex rumen microbial communities, 16S ribosomal RNA (rRNA) sequencing is widely used. Here, we evaluated the impact of bioinformatics pipelines on the observation of OTUs and taxonomic classification of 750 cattle rumen microbial samples by comparing three commonly used pipelines (LotuS, UPARSE, and QIIME) with Usearch. In LotuS-based analyses, 189 archaeal and 3894 bacterial OTUs were observed. The observed OTUs for the Usearch analysis were significantly larger than the LotuS results. We discovered 1495 OTUs for archaea and 92665 OTUs for bacteria using Usearch analysis. In addition, taxonomic assignments were made for the rumen microbial samples. All pipelines had consistent taxonomic annotations from the phylum to the genus level. A difference in relative abundance was calculated for all microbial levels, including Bacteroidetes (QIIME: 72.2%, Usearch: 74.09%), Firmicutes (QIIME: 18.3%, Usearch: 20.20%) for the bacterial phylum, Methanobacteriales (QIIME: 64.2%, Usearch: 45.7%) for the archaeal class, Methanobacteriaceae (QIIME: 35%, Usearch: 45.7%) and Methanomassiliicoccaceae (QIIME: 35%, Usearch: 31.13%) for archaeal family. However, the most prevalent archaeal class varied between these two annotation pipelines. The Thermoplasmata was the top class according to the QIIME annotation, whereas Methanobacteria was the top class according to Usearch.

Keywords: cattle rumen, rumen microbial, 16S rRNA gene sequencing, bioinformatics pipeline

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Authors: Janos Juhasz, Sandor Pongor, Balazs Ligeti

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Next-generation sequencing, especially whole genome shotgun sequencing, is becoming a common approach to gain insight into the microbiomes in a culture-independent way, even in clinical practice. It does not only give us information about the species composition of an environmental sample but opens the possibility to detect antimicrobial resistance and novel, or currently unknown, pathogens. Accurately and reliably detecting the microbial strains is a challenging task. Here we present a sensitive approach for detecting pathogens in metagenomics samples with special regard to detecting novel variants of known pathogens. We have developed a pipeline that uses fast, short read aligner programs (i.e., Bowtie2/BWA) and comprehensive nucleotide databases. Taxonomic binning is based on the lowest common ancestor (LCA) principle; each read is assigned to a taxon, covering the most significantly hit taxa. This approach helps in balancing between sensitivity and running time. The program was tested both on experimental and synthetic data. The results implicate that our method performs as good as the state-of-the-art BLAST-based ones, furthermore, in some cases, it even proves to be better, while running two orders magnitude faster. It is sensitive and capable of identifying taxa being present only in small abundance. Moreover, it needs two orders of magnitude less reads to complete the identification than MetaPhLan2 does. We analyzed an experimental anthrax dataset (B. anthracis strain BA104). The majority of the reads (96.50%) was classified as Bacillus anthracis, a small portion, 1.2%, was classified as other species from the Bacillus genus. We demonstrate that the evaluation of high-throughput sequencing data is feasible in a reasonable time with good classification accuracy.

Keywords: metagenomics, taxonomy binning, pathogens, microbiome, B. anthracis

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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: M. Kouyate, M. Sangare, S. Samake, S. Keita, H. G. Kim, D. H. Geschwind

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Background & Objectives: Human genetic studies can be expensive, even unaffordable, in developing countries, partly due to the sequencing costs. Our aim is to pilot the use of bioinformatics tools to guide scientifically valid, locally relevant, and economically sound autism genetic research in Mali. Methods: The following databases, NCBI, HGMD, and LSDB, were used to identify hot point mutations. Phenotype, transmission pattern, theoretical protein expression in the brain, the impact of the mutation on the 3D structure of the protein) were used to prioritize selected autism genes. We used the protein database, Modeller, and clustal W. Results: We found Mef2c (Gly27Ala/Leu38Gln), Pten (Thr131IIle), Prodh (Leu289Met), Nme1 (Ser120Gly), and Dhcr7 (Pro227Thr/Glu224Lys). These mutations were associated with endonucleases BseRI, NspI, PfrJS2IV, BspGI, BsaBI, and SpoDI, respectively. Gly27Ala/Leu38Gln mutations impacted the 3D structure of the Mef2c protein. Mef2c protein sequences across species showed a high percentage of similarity with a highly conserved MADS domain. Discussion: Mef2c, Pten, Prodh, Nme1, and Dhcr 7 gene mutation frequencies in the Malian population will be very informative. PCR coupled with restriction enzyme digestion can be used to screen the targeted gene mutations. Sanger sequencing will be used for confirmation only. This will cut down considerably the sequencing cost for gene-to-gene mutation screening. The knowledge of the 3D structure and potential impact of the mutations on Mef2c protein informed the protein family and altered function (ex. Leu38Gln). Conclusion & Future Work: Bio-informatics will positively impact autism research in Mali. Our approach can be applied to another neuropsychiatric disorder.

Keywords: bioinformatics, endonucleases, autism, Sanger sequencing, point mutations

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Authors: Mustafa M. Bob, Siti Izaidah Azmi, Mohd Hakim Ab Halim, Nur Syahida Abdul Jamal, Aznah Nor-Anuar, Zaini Ujang

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In this research, the formation and development of aerobic granular sludge (AGS) for domestic wastewater treatment application in hot climate conditions was studied using a sequencing batch reactor (SBR). The performance of the developed AGS in the removal of organic matter and nutrients from wastewater was also investigated. The operation of the reactor was based on the sequencing batch system with a complete cycle time of 3 hours that included feeding, aeration, settling, discharging and idling. The reactor was seeded with sludge collected from the municipal wastewater treatment plant in Madinah city, Saudi Arabia and operated at a temperature of 40ºC using synthetic wastewater as influent. Results showed that granular sludge was developed after an operation period of 30 days. The developed granular sludge had a good settling ability with the average size of the granules ranging from 1.03 to 2.42 mm. The removal efficiency of chemical oxygen demand (COD), ammonia nitrogen (NH3-N) and total phosphorus (TP) were 87.31%, 91.93% and 61.25% respectively. These results show that AGS can be developed at elevated temperatures and it is a promising technique to treat domestic wastewater in hot and low humidity climate conditions such as those encountered in Saudi Arabia.

Keywords: aerobic granular sludge, hot climate, sequencing batch reactor, domestic wastewater treatment

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Authors: Sumeet Rai, Anuj Tyagi, B. T. Naveen Kumar, Shubhkaramjeet Kaur, Niraj K. Singh

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Since their discovery, indiscriminate use of antibiotics in human, veterinary and aquaculture systems has resulted in global emergence/spread of multidrug-resistant bacterial pathogens. Thus, the need for alternative approaches to control bacterial infections has become utmost important. High selectivity/specificity of bacteriophages (phages) permits the targeting of specific bacteria without affecting the desirable flora. In this study, a lytic phage (Ahp1) specific to Aeromonas hydrophila subsp. hydrophila was isolated from finfish aquaculture pond. The host range of Ahp1 range was tested against 10 isolates of A. hydrophila, 7 isolates of A. veronii, 25 Vibrio cholerae isolates, 4 V. parahaemolyticus isolates and one isolate each of V. harveyi and Salmonella enterica collected previously. Except the host A. hydrophila subsp. hydrophila strain, no lytic activity against any other bacterial was detected. During the adsorption rate and one-step growth curve analysis, 69.7% of phage particles were able to get adsorbed on host cell followed by the release of 93 ± 6 phage progenies per host cell after a latent period of ~30 min. Phage nucleic acid was extracted by column purification methods. After determining the nature of phage nucleic acid as dsDNA, phage genome was subjected to next-generation sequencing by generating paired-end (PE, 2 x 300bp) reads on Illumina MiSeq system. De novo assembly of sequencing reads generated circular phage genome of 42,439 bp with G+C content of 58.95%. During open read frame (ORF) prediction and annotation, 22 ORFs (out of 49 total predicted ORFs) were functionally annotated and rest encoded for hypothetical proteins. Proteins involved in major functions such as phage structure formation and packaging, DNA replication and repair, DNA transcription and host cell lysis were encoded by the phage genome. The complete genome sequence of Ahp1 along with gene annotation was submitted to NCBI GenBank (accession number MF683623). Stability of Ahp1 preparations at storage temperatures of 4 °C, 30 °C, and 40 °C was studied over a period of 9 months. At 40 °C storage, phage counts declined by 4 log units within one month; with a total loss of viability after 2 months. At 30 °C temperature, phage preparation was stable for < 5 months. On the other hand, phage counts decreased by only 2 log units over a period of 9 during storage at 4 °C. As some of the phages have also been reported as glycerol sensitive, the stability of Ahp1 preparations in (0%, 15%, 30% and 45%) glycerol stocks were also studied during storage at -80 °C over a period of 9 months. The phage counts decreased only by 2 log units during storage, and no significant difference in phage counts was observed at different concentrations of glycerol. The Ahp1 phage discovered in our study had a very narrow host range and it may be useful for phage typing applications. Moreover, the endolysin and holin genes in Ahp1 genome could be ideal candidates for recombinant cloning and expression of antimicrobial proteins.

Keywords: Aeromonas hydrophila, endolysin, phage, narrow host range

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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: Eyup Ozkan, Ozkan U. Nalbantoglu, Aycan Gundogdu, Mehmet Hora, A. Emre Onuk

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The human microbiome has been associated with cardiological conditions and this relationship is becoming to be defined beyond the gastrointestinal track. In this study, we investigate the alteration in circulatory microbiota in the context of Coronary Artery Disease (CAD). We received circulatory blood samples from suspected CAD patients and maintain 16S ribosomal RNA sequencing to identify each patient’s microbiome. It was found that Corynebacterium and Methanobacteria genera show statistically significant differences between healthy and CAD patients. The overall biodiversities between the groups were observed to be different revealed by machine learning classification models. We also achieve and demonstrate the performance of a diagnostic method using circulatory blood microbiome-based estimation.

Keywords: coronary artery disease, blood microbiome, machine learning, angiography, next-generation sequencing

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Authors: Mohammed Ali

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A flexible manufacturing system has been studied under different manufacturing strategies. The aim of this paper is to test the impact of number of pallets and routing flexibility (design strategy) on system performance operating at different sequencing and dispatching rules (control strategies) at unbalanced load condition (planning strategies). A computer simulation model is developed to evaluate the effects of aforementioned strategies on the make-span time, which is taken as the system performance measure. The impact of number of pallets is shown with the different levels of routing flexibility. In this paper, the same manufacturing system is modeled under different combination of sequencing and dispatching rules. The result of the simulation shows that there is definite range of pallets for each level of routing flexibility at which the systems performs satisfactorily.

Keywords: flexible manufacturing system, manufacturing, strategy, makespan

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Authors: Hammad Khan, Wookeun Bae

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Major concerns regarding nitritation of low-strength ammonium wastewaters include low ammonium loading rates (usually below 0.2 kg/m3-d) and uncertainty about long-term stability of the process. The purpose of this study was to test a sequencing batch reactor (SBR) filled with cell-immobilized polyethylene glycol (PEG) pellets to see if it could achieve efficient and stable nitritation under various environmental conditions. SBR was fed with synthetic ammonium wastewater of 30±2 mg-N/L and pH: 8±0.05, maintaining the dissolved oxygen concentration of 1.7±0.2 mg/L and the temperature at 30±1oC. The reaction was easily converted to partial nitrification mode within a month by feeding relatively high ammonium substrate (~100 mg-N/L) in the beginning. We observed stable nitritation over 300 days with high ammonium loading rates (as high as ~1.1 kg-N/m3-d), nitrite accumulation rates (mostly over 97%) and ammonium removal rate (mostly over 95%). DO was a major limiting substrate when the DO concentration was below ~4 mg/L and the NH4+-N concentration was above 5 mg/L, giving almost linear increase in the ammonium oxidation rate with the bulk DO increase. Low temperatures mainly affected the reaction rate, which could be compensated for by increasing the pellet volume (i.e. biomass). Our results demonstrated that an SBR filled with small cell-immobilized PEG pellets could achieve very efficient and stable nitritation of a low-strength ammonium wastewater.

Keywords: ammonium loading rate (ALR), cell-immobilization, long-term nitritation, sequencing batch reactor (SBR), sewage treatment

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Authors: Alisa Kazarina, Guntis Gerhards, Elina Petersone-Gordina, Ilva Pole, Viktorija Igumnova, Janis Kimsis, Valentina Capligina, Renate Ranka

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Human body is inhabited by a vast number of microorganisms, collectively known as the human microbiome, and there is a tremendous interest in evolutionary changes in human microbial ecology, diversity and function. The field of paleomicrobiology, study of ancient human microbiome, is powered by modern techniques of Next Generation Sequencing (NGS), which allows extracting microbial genomic data directly from archaeological sample of interest. One of the major techniques is 16S rRNA gene sequencing, by which certain 16S rRNA gene hypervariable regions are being amplified and sequenced. However, some limitations of this method exist including the taxonomic precision and efficacy of different regions used. The aim of this study was to evaluate the phylogenetic sensitivity of different 16S rRNA gene hypervariable regions for microbiome studies in the archaeological samples. Towards this aim, archaeological bone samples and corresponding soil samples from each burial environment were collected in Medieval cemeteries in Latvia. The Ion 16S™ Metagenomics Kit targeting different 16S rRNA gene hypervariable regions was used for library construction (Ion Torrent technologies). Sequenced data were analysed by using appropriate bioinformatic techniques; alignment and taxonomic representation was done using Mothur program. Sequences of most abundant genus were further aligned to E. coli 16S rRNA gene reference sequence using MEGA7 in order to identify the hypervariable region of the segment of interest. Our results showed that different hypervariable regions had different discriminatory power depending on the groups of microbes, as well as the nature of samples. On the basis of our results, we suggest that wider range of primers used can provide more accurate recapitulation of microbial communities in archaeological samples. Acknowledgements. This work was supported by the ERAF grant Nr. 1.1.1.1/16/A/101.

Keywords: 16S rRNA gene, ancient human microbiome, archaeology, bioinformatics, genomics, microbiome, molecular biology, next-generation sequencing

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Authors: Cliff Shunsheng Han

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Identifying the direct allergy cause that can be easily mitigated is the foundation to stop the allergy epidemic that has been started in the seventies. It has confirmed that the personal and social hygiene practices are associated with the allergy prevalence. But direct causes have been found, and proposed translational measures have not been effective. This study, assisted by a particular case of allergies, has seen the direct cause of allergies, developed a valid test resulted in lasting relief for allergies, and constructed theory describing general relationship between microbiota and host immune system. Saliva samples were collected from a subject for three years during which time the person experienced yearlong allergy, seasonal allergy, and remission of allergy symptoms. Bacterial DNA was extracted and 16S rRNA genes were profiled with Illumina sequencing technology. The analyzing results indicate that the possible direct cause of allergy is the lacking probiotic bacteria in the oral cavity, such as genera Streptococcus and Veilonella, that can produce metabolites to pacify immune system. Targeted promotion of those bacteria with a compound designed for them, has led to lasting remissions of allergic rhinitis. During the development of the translational measure, the subject's oral biofilm was completely destructed by a moderate fever due to an unrelated respiratory infection. The incident not only facilitated the development of the heat based microbiota reseeding procedure but also indicated a possible natural switch that subsequently increases the efficacy of the immune system previously restrained by metabolites from microbiota. These results lead to the proposal of a Theory of Negative Trigger (TNT) to describe the relationship between oral probiotics and immune system, in which probiotics are the negative trigger that will release the power of immune system when removed by fever or modern lifestyles. This study could open doors leading to further understanding of how the immune system functions under the influence of microbiota as well as validate simple traditional practices for healthy living.

Keywords: oral microbiome, allergy, immune system, infection

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Authors: Cheng-Yang Lee, Petrus Tang, Tzu-Hao Chang

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Copy number variations (CNVs) have played an important role in many kinds of human diseases, such as Autism, Schizophrenia and a number of cancers. Many diseases are found in genome coding regions and whole exome sequencing (WES) is a cost-effective and powerful technology in detecting variants that are enriched in exons and have potential applications in clinical setting. Although several algorithms have been developed to detect CNVs using WES and compared with other algorithms for finding the most suitable methods using their own samples, there were not consistent datasets across most of algorithms to evaluate the ability of CNV detection. On the other hand, most of algorithms is using command line interface that may greatly limit the analysis capability of many laboratories. We create a series of simulated WES datasets from UCSC hg19 chromosome 22, and then evaluate the CNV detective ability of 19 algorithms from OMICtools database using our simulated WES datasets. We compute the sensitivity, specificity and accuracy in each algorithm for validation of the exome-derived CNVs. After comparison of 19 algorithms from OMICtools database, we construct a platform to install all of the algorithms in a virtual machine like VirtualBox which can be established conveniently in local computers, and then create a simple script that can be easily to use for detecting CNVs using algorithms selected by users. We also build a table to elaborate on many kinds of events, such as input requirement, CNV detective ability, for all of the algorithms that can provide users a specification to choose optimum algorithms.

Keywords: whole exome sequencing, copy number variations, omictools, pipeline

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Authors: Chia-Jung Li, Li-Te Lin, Kuan-Hao Tsui

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The study identifies SPP1 as a potential gene associated with ovarian aging, revealing a significant decline in its expression in aged ovaries. SPP1, also known as osteopontin (OPN), is a multifunctional glycoprotein involved with regulatory proteins and pro-inflammatory immune chemokines. However, its genetic links to ovarian aging have not been extensively explored. Spatial transcriptomic analyses were conducted on ovaries from young and aged female mice, along with a sample from a 73-year-old individual. Additionally, single-cell RNA sequencing analysis was performed to identify associations between SPP1 and key genes. The study focused on crucial genes, including ITGAV, ITGB1, CD44, MMP3, and FN1, with a particular emphasis on the correlation between SPP1 and ITGB1. The findings indicate a significant decline in SPP1 expression in aged ovaries, which was consistent in the 73-year-old sample. Single-cell RNA sequencing unveiled associations between SPP1 and key genes, emphasizing a strong co-expression correlation between SPP1 and ITGB1. While the study provides valuable insights, further research is necessary to understand the broader implications and potential applications of SPP1 in ovarian aging. Translating these findings to clinical settings requires careful consideration. The identification of SPP1 as a gene implicated in ovarian aging opens new avenues for advancing precision medicine and refining treatment strategies for conditions related to ovarian aging.

Keywords: SPP1, ovarian aging, spatial transcriptomic, single-cell RNA sequencing

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Authors: Elizabeth Starzycka-Korbas, Michal Starzycki, Wojciech Rybinski, Mirosława Dabert

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For a few years, the populations of pathogenic fungi occurring in winter oilseed rape in Malyszyn were analyzed. Brassica napus L. in Poland and in the world is a source of energy for both the men (oil), and animals, as post-extraction middling, as well as a motor fuel (oil, biofuel) therefore studies of this type are very important. The species composition of pathogenic fungi can be an indicator of seed yield. The occurrence of oilseed rape pathogens during several years were analyzed using the sequencing method DNA ITS. The results were compared in the gene bank using the program NCBI / BLAST. In field conditions before harvest of oilseed rape presence of pathogens infesting B. napus has been assessed. For example, in 2015, 150 samples have been isolated and applied to PDA medium for the identification of belonging species. From all population has been selected mycelium of 83 isolates which were sequenced. Others (67 isolates) were pathogenic fungi of the genus Alternaria which are easily to recognize. The population of pathogenic species on oilseed rape have been identified after analyzing the DNA ITS and include: Leptosphaeria sp. 38 (L. maculans 25, L. biglobosa 13), Alternaria sp. 29, Fusarium sp. 3, Sclerotinia sclerotiorum 7, heterogeneous 6, total of 83 isolates. The genus Alternaria sp. fungi wear the largest share of B. napus pathogens in particular years. Another dangerous species for oilseed rape was Leptosphaeria sp. Populations of pathogens in each year were different. The number of pathogens occurring in the field and their composition is very important for breeders and farmers because of the possible selection of the most resistant genotypes for sowing in the next growing season.

Keywords: B. napus, DNA ITS Sequencing, pathogenic fungi, population

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Authors: Il Ho Park, Joong Seob Lee, Sung Hun Kang, Jae-Min Shin, Il Seok Park, Seok Min Hong, Seok Jin Hong

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Introduction: The human microbiota is the aggregate of microorganisms, and the bacterial microbiome of the human digestive tract contributes to both health and disease. In health, bacteria are key components in the development of mucosal barrier function and in innate and adaptive immune responses, and they also work to suppress the establishment of pathogens. In human upper airway, the sinonasal microbiota might play an important role in chronic rhinosinusitis (CRS). The purpose of this study is to investigate the human upper airway microbiome in CRS patients and to compare the sinonasal microbiome of adults with children. Materials and methods: A total of 19 samples from 19 patients (Group1; 9 CRS in children, aged 5 to 14 years versus Group 2; 10 CRS in adults aged 21 to 59 years) were examined. Swabs were collected from the middle meatus and/or anterior ethmoid region under general anesthesia during endoscopic sinus surgery or tonsillectomy. After DNA extraction from swab samples, we analysed bacterial microbiome consortia using 16s rRNA gene sequencing approach (the Illumina MiSeq platform). Results: In this study, relatively abundance of the six bacterial phyla and tremendous genus and species found in substantial amounts in the individual sinus swab samples, include Corynebacterium, Hemophilus, Moraxella, and Streptococcus species. Anaerobes like Fusobacterium and Bacteroides were abundantly present in the children group, Bacteroides and Propionibacterium were present in adults group. In genus, Haemophilus was the most common CRS microbiome in children and Corynebacterium was the most common CRS microbiome in adults. Conclusions: Our results show the diversity of human upper airway microbiome, and the findings will suggest that CRS is a polymicrobial infection. The Corynebacterium and Hemophilus may live as commensals on mucosal surfaces of sinus in the upper respiratory tract. The further study will be needed for analysis of microbiome-human interactions in upper airway and CRS.

Keywords: microbiome, upper airway, chronic rhinosinusitis, adult and children

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Authors: Tarek Aboueldahab, Hanan Farag

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Parallel Job Shop Scheduling Problem (JSP) is a multi-objective and multi constrains NP- optimization problem. Traditional Artificial Intelligence techniques have been widely used; however, they could be trapped into the local minimum without reaching the optimum solution, so we propose a hybrid Artificial Intelligence model (AI) with Discrete Breeding Swarm (DBS) added to traditional Artificial Intelligence to avoid this trapping. This model is applied in the cost minimization of the Car Sequencing and Operator Allocation (CSOA) problem. The practical experiment shows that our model outperforms other techniques in cost minimization.

Keywords: parallel job shop scheduling problem, artificial intelligence, discrete breeding swarm, car sequencing and operator allocation, cost minimization

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Authors: Lohendy Munoz-Vargas, Stephen O. Opiyo, Rose Digianantonio, Michele L. Williams, Asela Wijeratne, Gregory Habing

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Non-typhoidal Salmonella enterica is a zoonotic pathogen with critical importance in animal and public health. The persistence of Salmonella on farms affects animal productivity and health, and represents a risk for food safety. The intestinal microbiota plays a fundamental role in the colonization and invasion of this ubiquitous microorganism. To overcome the colonization resistance imparted by the gut microbiome, Salmonella uses invasion strategies and the host inflammatory response to survive, proliferate, and establish infections with diverse clinical manifestations. Cattle serve as reservoirs of Salmonella, and periparturient cows have high prevalence of Salmonella shedding; however, to author`s best knowledge, little is known about the association between the gut microbiome and the onset of Salmonella shedding during the periparturient period. Thus, the objective of this study was to assess the association between changes in bacterial communities and the onset of Salmonella shedding in cattle approaching parturition. In a prospective cohort study, fecal samples from 98 dairy cows originating from four different farms were collected at four time points relative to calving (-3 wks, -1 wk, +1 wk, +3 wks). All 392 samples were cultured for Salmonella. Sequencing of the V4 region of the 16S rRNA gene using the Illumina platform was completed to evaluate the fecal microbiome in a selected sample subset. Analyses of microbial composition, diversity, and structure were performed according to time points, farm, and Salmonella onset status. Individual cow fecal microbiomes, predominated by Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria phyla, significantly changed before and after parturition. Microbial communities from different farms were distinguishable based on multivariate analysis. Although there were significant differences in some bacterial taxa between Salmonella positive and negative samples, our results did not identify differences in the fecal microbial diversity or structure for cows with and without the onset of Salmonella shedding. These data suggest that determinants other than the significant changes in the fecal microbiome influence the periparturient onset of Salmonella shedding in dairy cattle.

Keywords: dairy cattle, microbiome, periparturient, Salmonella

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Authors: Vanessa De Carvalho, Chad MacPherson, Julien Tremblay, Julie Champagne, Stephanie-Anne Girard

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Background: The interactions between bacteria and fungi in the human vaginal microbiome are fundamental to the concept of health and disease. The means by which the microbiota and mycobiota interact is still poorly understood and further studies are necessary to properly characterize this complex ecosystem. The aim of this study was to select a DNA extraction method capable of recovering high qualities of fungal and bacterial DNA from a single vaginal swab. Methods: 11 female volunteers ( ≥ 20 to < 55 years old) self-collected vaginal swabs in triplicates. Three commercial extraction kits: Masterpure Yeast Purification kit (Epicenter), PureLink™ Microbiome DNA Purification kit (Invitrogen), and Quick-DNA™ Fecal/Soil Microbe Miniprep kit (Zymo) were evaluated on the ability to recover fungal and bacterial DNA simultaneously. The extraction kits were compared on the basis of recovery, yield, purity, and the community richness of bacterial (16S rRNA - V3-V4 region) and fungal (ITS1) microbiota composition by Illumina MiSeq amplicon sequencing. Results: Recovery of bacterial DNA was achieved with all three kits while fungal DNA was only consistently recovered with Masterpure Yeast Purification kit (yield and purity). Overall, all kits displayed similar microbiota profiles for the top 20 OTUs; however, Quick-DNA™ Fecal/Soil Microbe Miniprep kit (Zymo) showed more species richness than the other two kits. Conclusion: In the present study, Masterpure Yeast purification kit proved to be a good candidate for purification of high quality fungal and bacterial DNA simultaneously. These findings have potential benefits that could be applied in future vaginal microbiome research. Whilst the use of a single extraction method would lessen the burden of multiple swab sampling, decrease laboratory workload and off-set costs associated with multiple DNA extractions, thoughtful consideration must be taken when selecting an extraction kit depending on the desired downstream application.

Keywords: bacterial vaginosis, DNA extraction, microbiota, mycobiota, vagina, vulvovaginal candidiasis, women’s health

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Authors: Ali Alattabi, Clare Harris, Rafid Alkhaddar, Ali Alzeyadi

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Petroleum refinery wastewater (PRW) can be considered as one of the most significant source of aquatic environmental pollution. It consists of oil and grease along with many other toxic organic pollutants. In recent years, a new technique was implemented using different types of membranes and sequencing batch reactors (SBRs) to treat PRW. SBR is a fill and draw type sludge system which operates in time instead of space. Many researchers have optimised SBRs’ operating conditions to obtain maximum removal of undesired wastewater pollutants. It has gained more importance mainly because of its essential flexibility in cycle time. It can handle shock loads, requires less area for operation and easy to operate. However, bulking sludge or discharging floating or settled sludge during the draw or decant phase with some SBR configurations are still one of the problems of SBR system. The main aim of this study is to develop and innovative design for the SBR optimising the process variables to result is a more robust and efficient process. Several experimental tests will be developed to determine the removal percentages of chemical oxygen demand (COD), Phenol and nitrogen compounds from synthetic PRW. Furthermore, the dissolved oxygen (DO), pH and oxidation-reduction potential (ORP) of the SBR system will be monitored online to ensure a good environment for the microorganisms to biodegrade the organic matter effectively.

Keywords: petroleum refinery wastewater, sequencing batch reactor, hydraulic retention time, Phenol, COD, mixed liquor suspended solids (MLSS)

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Authors: Mohammed Ali

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In this paper, evaluation of flexible manufacturing system is made under different manufacturing strategies. The objective of this paper is to test the impact of pallets and routing flexibility on system performance operating at different sequencing rules, dispatching rules and at unbalanced load condition. A computer simulation model is developed to evaluate the effects of aforementioned manufacturing strategies on the make-span performance of flexible manufacturing system. The impact of number of pallets is shown with the different levels of routing flexibility. In this paper, the same manufacturing system is modeled under different combination of sequencing and dispatching rules. A series of simulation experiments are conducted and results analyzed. The result of the simulation shows that there is impact of pallets and routing flexibility on the performance of the system.

Keywords: flexibility, flexible manufacturing system, pallets, make-span, simulation

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Authors: Irena Maus, Katharina Gabriella Cibis, Andreas Bremges, Yvonne Stolze, Geizecler Tomazetto, Daniel Wibberg, Helmut König, Alfred Pühler, Andreas Schlüter

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Within the agricultural sector, the production of biogas from organic substrates represents an economically attractive technology to generate bioenergy. Complex consortia of microorganisms are responsible for biomass decomposition and biogas production. Recently, species belonging to the phylum Thermotogae were detected in thermophilic biogas-production plants utilizing renewable primary products for biomethanation. To analyze adaptive genome features of representative Thermotogae strains, Defluviitoga tunisiensis L3 was isolated from a rural thermophilic biogas plant (54°C) and completely sequenced on an Illumina MiSeq system. Sequencing and assembly of the D. tunisiensis L3 genome yielded a circular chromosome with a size of 2,053,097 bp and a mean GC content of 31.38%. Functional annotation of the complete genome sequence revealed that the thermophilic strain L3 encodes several genes predicted to facilitate growth of this microorganism on arabinose, galactose, maltose, mannose, fructose, raffinose, ribose, cellobiose, lactose, xylose, xylan, lactate and mannitol. Acetate, hydrogen (H2) and carbon dioxide (CO2) are supposed to be end products of the fermentation process. The latter gene products are metabolites for methanogenic archaea, the key players in the final step of the anaerobic digestion process. To determine the degree of relatedness of dominant biogas community members within selected digester systems to D. tunisiensis L3, metagenome sequences from corresponding communities were mapped on the L3 genome. These fragment recruitments revealed that metagenome reads originating from a thermophilic biogas plant covered 95% of D. tunisiensis L3 genome sequence. In conclusion, availability of the D. tunisiensis L3 genome sequence and insights into its metabolic capabilities provide the basis for biotechnological exploitation of genome features involved in thermophilic fermentation processes utilizing renewable primary products.

Keywords: genome sequence, thermophilic biogas plant, Thermotogae, Defluviitoga tunisiensis

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Authors: Huajuan Shi

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Background: The biopsy of the preimplantation embryo may increase the potential risk and concern of embryo viability. Clinically discarded spent embryo medium (SEM) has entered the view of researchers, sparking an interest in noninvasive embryo screening. However, one of the major restrictions is the extremelty low quantity of cf-RNA, which is difficult to efficiently and unbiased amplify cf-RNA using traditional methods. Hence, there is urgently need to an efficient and low bias amplification method which can comprehensively and accurately obtain cf-RNA information to truly reveal the state of SEM cf-RNA. Result: In this present study, we established an agarose PCR amplification system, and has significantly improved the amplification sensitivity and efficiency by ~90 fold and 9.29 %, respectively. We applied agarose to sequencing library preparation (named AG-seq) to quantify and characterize cf-RNA in SEM. The number of detected cf-RNAs (3533 vs 598) and coverage of 3' end were significantly increased, and the noise of low abundance gene detection was reduced. The increasing percentage 5' end adenine and alternative splicing (AS) events of short fragments (< 400 bp) were discovered by AG-seq. Further, the profiles and characterizations of cf-RNA in spent cleavage medium (SCM) and spent blastocyst medium (SBM) indicated that 4‐mer end motifs of cf-RNA fragments could remarkably differentiate different embryo development stages. Significance: This study established an efficient and low-cost SEM amplification and library preparation method. Not only that, we successfully described the characterizations of SEM cf-RNA of preimplantation embryo by using AG-seq, including abundance features fragment lengths. AG-seq facilitates the study of cf-RNA as a noninvasive embryo screening biomarker and opens up potential clinical utilities of trace samples.

Keywords: cell-free RNA, agarose, spent embryo medium, RNA sequencing, non-invasive detection

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Authors: Mpho Mokoatle, Darlington Mapiye, James Mashiyane, Stephanie Muller, Gciniwe Dlamini

Abstract:

Great advances in high-throughput sequencing technologies have resulted in availability of huge amounts of sequencing data in public and private repositories, enabling a holistic understanding of complex biological phenomena. Sequence data are used for a wide range of applications such as gene annotations, expression studies, personalized treatment and precision medicine. However, this rapid growth in sequence data poses a great challenge which calls for novel data processing and analytic methods, as well as huge computing resources. In this work, a machine and statistical learning approach for DNA sequence classification based on $k$-mer representation of sequence data is proposed. The approach is tested using whole genome sequences of Mycobacterium tuberculosis (MTB) isolates to (i) reduce the size of genomic sequence data, (ii) identify an optimum size of k-mers and utilize it to build classification models, (iii) predict the phenotype from whole genome sequence data of a given bacterial isolate, and (iv) demonstrate computing challenges associated with the analysis of whole genome sequence data in producing interpretable and explainable insights. The classification models were trained on 104 whole genome sequences of MTB isoloates. Cluster analysis showed that k-mers maybe used to discriminate phenotypes and the discrimination becomes more concise as the size of k-mers increase. The best performing classification model had a k-mer size of 10 (longest k-mer) an accuracy, recall, precision, specificity, and Matthews Correlation coeffient of 72.0%, 80.5%, 80.5%, 63.6%, and 0.4 respectively. This study provides a comprehensive approach for resampling whole genome sequencing data, objectively selecting a k-mer size, and performing classification for phenotype prediction. The analysis also highlights the importance of increasing the k-mer size to produce more biological explainable results, which brings to the fore the interplay that exists amongst accuracy, computing resources and explainability of classification results. However, the analysis provides a new way to elucidate genetic information from genomic data, and identify phenotype relationships which are important especially in explaining complex biological mechanisms.

Keywords: AWD-LSTM, bootstrapping, k-mers, next generation sequencing

Procedia PDF Downloads 167

Authors: Darlington Mapiye, Mpho Mokoatle, James Mashiyane, Stephanie Muller, Gciniwe Dlamini

Abstract:

Great advances in high-throughput sequencing technologies have resulted in availability of huge amounts of sequencing data in public and private repositories, enabling a holistic understanding of complex biological phenomena. Sequence data are used for a wide range of applications such as gene annotations, expression studies, personalized treatment and precision medicine. However, this rapid growth in sequence data poses a great challenge which calls for novel data processing and analytic methods, as well as huge computing resources. In this work, a machine and statistical learning approach for DNA sequence classification based on k-mer representation of sequence data is proposed. The approach is tested using whole genome sequences of Mycobacterium tuberculosis (MTB) isolates to (i) reduce the size of genomic sequence data, (ii) identify an optimum size of k-mers and utilize it to build classification models, (iii) predict the phenotype from whole genome sequence data of a given bacterial isolate, and (iv) demonstrate computing challenges associated with the analysis of whole genome sequence data in producing interpretable and explainable insights. The classification models were trained on 104 whole genome sequences of MTB isoloates. Cluster analysis showed that k-mers maybe used to discriminate phenotypes and the discrimination becomes more concise as the size of k-mers increase. The best performing classification model had a k-mer size of 10 (longest k-mer) an accuracy, recall, precision, specificity, and Matthews Correlation coeffient of 72.0 %, 80.5 %, 80.5 %, 63.6 %, and 0.4 respectively. This study provides a comprehensive approach for resampling whole genome sequencing data, objectively selecting a k-mer size, and performing classification for phenotype prediction. The analysis also highlights the importance of increasing the k-mer size to produce more biological explainable results, which brings to the fore the interplay that exists amongst accuracy, computing resources and explainability of classification results. However, the analysis provides a new way to elucidate genetic information from genomic data, and identify phenotype relationships which are important especially in explaining complex biological mechanisms

Keywords: AWD-LSTM, bootstrapping, k-mers, next generation sequencing

Procedia PDF Downloads 159

Authors: Swarkar Sharma, Ekta Rai, Ankit Mahajan, Parvinder Kumar, Manoj K Dhar, Sushil Razdan, Kumarasamy Thangaraj, Carol Wise, Shiro Ikegawa M.D., K.K. Pandita M.D.

Abstract:

Rare disorders are poorly understood hence, remain uncharacterized or patients are misdiagnosed and get poor medical attention. A rare mysterious skeletal disorder that remained unidentified for decades and rendered many people physically challenged and disabled for life has been reported in an isolated remote village ‘Arai’ of Poonch district of Jammu and Kashmir. This village is located deep in mountains and the population residing in the region is highly consanguineous. In our survey of the region, 70 affected people were reported, showing similar phenotype, in the village with a population of approximately 5000 individuals. We were able to collect samples from two multi generational extended families from the village. Through Whole Exome sequencing (WES), we identified a rare variation NM_003880.3:c.156C>A NP_003871.1:p.Cys52Ter, which results in introduction of premature stop codon in WISP3 gene. We found this variation perfectly segregating with the disease in one of the family. However, this variation was absent in other family. Interestingly, a novel splice site mutation at position c.643+1G>A of WISP3 gene, perfectly segregating with the disease was observed in the second family. Thus, exploiting WES and putting different evidences together (familial histories and genetic data, clinical features, radiological and biochemical tests and findings), the disease has finally been diagnosed as a very rare recessive hereditary skeletal disease “Progressive Pseudorheumatoid Arthropathy of Childhood” (PPAC) also known as “Spondyloepiphyseal Dysplasia Tarda with Progressive Arthropathy” (SEDT-PA). This genetic characterization and identification of the disease causing mutations will aid in genetic counseling, critically required to curb this rare disorder and to prevent its appearance in future generations in the population. Further, understanding of the role of WISP3 gene the biological pathways should help in developing treatment for the disorder.

Keywords: whole exome sequencing, Next Generation Sequencing, rare disorders

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Authors: Ayman K. El Essawy, Amal M. Hosny, Hala M. Abu Shady

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The rapid detection of TB and drug resistance, both optimizes treatment and improves outcomes. In the current study, respiratory specimens were collected from 155 patients. Conventional susceptibility testing and MIC determination were performed for rifampicin (RIF) and isoniazid (INH). Genotype MTBDRplus assay, which is a molecular genetic assay based on the DNA-STRIP technology and specific gene sequencing with primers for rpoB, KatG, and mab-inhA genes were used to detect mutations associated with resistance to rifampicin and isoniazid. In comparison to other categories, most of rifampicin resistant (61.5%) and isoniazid resistant isolates (47.1%) were from patients relapsed in treatment. The genotypic profile (using Genotype MTBDRplus assay) of multi-drug resistant (MDR) isolates showed missing of katG wild type 1 (WT1) band and appearance of mutation band katG MUT2. For isoniazid mono-resistant isolates, 80% showed katG MUT1, 20% showed katG MUT1, and inhA MUT1, 20% showed only inhA MUT1. Accordingly, 100% of isoniazid resistant strains were detected by this assay. Out of 17 resistant strains, 16 had mutation bands for katG distinguished high resistance to isoniazid. The assay could clearly detect rifampicin resistance among 66.7% of MDR isolates that showed mutation band rpoB MUT3 while 33.3% of them were considered as unknown. One mono-resistant rifampicin isolate did not show rifampicin mutation bands by Genotype MTBDRplus assay, but it showed an unexpected mutation in Codon 531 of rpoB by DNA sequence analysis. Rifampicin resistance in this strain could be associated with a mutation in codon 531 of rpoB (based on molecular sequencing), and Genotype MTBDRplus assay could not detect the associated mutation. If the results of Genotype MTBDRplus assay and sequencing were combined, this strain shows hetero-resistance pattern. Gene sequencing of eight selected isolates, previously tested by Genotype MTBDRplus assay, could detect resistance mutations mainly in codon 315 (katG gene), position -15 in inhA promotes gene for isoniazid resistance and codon 531 (rpoB gene) for rifampicin resistance. Genotyping techniques allow distinguishing between recurrent cases of reinfection or reactivation and supports epidemiological studies.

Keywords: M. tuberculosis, rpoB, KatG, inhA, genotype MTBDRplus

Procedia PDF Downloads 166