Search results for: complete genome sequencing

Authors: Busra Mutlu Ipek, Merve Mutlu, Ahmet Mutlu

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Novel coronavirus COVID-19, which has recently influenced the world, poses a great threat to humanity. In order to overcome this challenging situation, scientists are working on developing effective vaccine against coronavirus. Many experts and researchers have also produced articles and done studies on this highly important subject. In this direction, this special topic was chosen for article to make a contribution to this area. The purpose of this article is to perform RNA sequence analysis of selected virus forms in the Coronaviridae family and predict/classify SARS-CoV-2 (COVID-19) from other selected complete genomes in coronaviridae family using proposed Artificial Neural Network(ANN) algorithm.

Keywords: Coronaviridae family, COVID-19, RNA sequencing, ANN, neural network

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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: 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: Zhao Yang Liu, Jing Tao

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The Asian long-horned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae: Lamiinae), is a wood-borer and polyphagous xylophages native to Asia and killing healthy trees. As it causes serious danger to trees, the beetle has been paid close attention in the world. However, the genetic markers limited, especially microsatellite. In this study, 24 novel simple sequence repeat (SSR) molecular markers, a powerful tool for genetic diversity studies and linkage map construction, were developed and characterized from whole genome shotgun sequences. We developed SSR loci of 2 to 6 repeated and perfect units including 9895 points, the density of SSRs was found one SSR per 56.57 kb and the abundance of SSR was 0.02/kb, besides 140 types of repeats motifs were found. Half of the 48 pairs SSR primers (containing 4 di-, 7 tri-, 2 tetra- and 11 hexamers SSRs) we selected randomly from 1222 pairs of primers were polymorphism. The number of alleles for these markers in 48 individuals varied from 3 to 21 with an average of 7.71, the number of effective alleles ranged from 1.22 to 9.97 with an average of 3.54. Besides this, the polymorphic information content (PIC) ranged from 0.18 to 0.89 with a mean of 0.65, And Shannon's Information index (I) ranged from 0.46 to 2.62 with an average of 1.44. The results suggest that the method for screening of SSR in the whole genome is feasible and efficient. SSR markers developed in this study can be used for population genetic studies of A. glabripennis. Moreover, they may also be helpful for the development of microsatellites for other Coleoptera.

Keywords: SSR markers, Anoplophora glabripennis, genetic diversity, whole genome

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Authors: Shagufta Jabeen, Faez Jesse Firdaus Abdullah, Zunita Zakaria, Nurulfiza Mat Isa, Yung Chie Tan, Wai Yan Yee, Abdul Rahman Omar

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Pasteurella multocida is associated with acute, as well as, chronic infections in avian and bovine such as pasteurellosis and hemorrhagic septicemia (HS) in cattle and buffaloes. Iron is one of the most important nutrients for pathogenic bacteria including Pasteurella and acts as a cofactor or prosthetic group in several essential enzymes and is needed for amino acid, pyrimidine, and DNA biosynthesis. In our recent study, we showed that 2% of Pasteurella multocida serotype A strain PMTB2.1 encode for iron regulating genes (Accession number CP007205.1). Genome sequencing of other Pasteurella multocida serotypes namely PM70 and HB01 also indicated up to 2.5% of the respective genome encode for iron regulating genes, suggesting that Pasteurella multocida genome comprises of multiple systems for iron uptake. Since P. multocida PMTB2.1 has more than 40 CDs out of 2097 CDs (approximately 2%), encode for iron-regulated. The gene expression profiling of four iron-regulating genes namely fbpb, yfea, fece and fur were characterized under iron-restricted environment. The P. multocida strain PMTB2.1 was grown in broth with and without iron chelating agent and samples were collected at different time points. Relative mRNA expression profile of these genes was determined using Taqman probe based real-time PCR assay. The data analysis, normalization with two house-keeping genes and the quantification of fold changes were carried out using Bio-Rad CFX manager software version 3.1. Results of this study reflect that iron reduced environment has significant effect on expression profile of iron regulating genes (p < 0.05) when compared to control (normal broth) and all evaluated genes act differently with response to iron reduction in media. The highest relative fold change of fece gene was observed at early stage of treatment indicating that PMTB2.1 may utilize its periplasmic protein at early stage to acquire iron. Furthermore, down-regulation expression of fece with the elevated expression of other genes at later time points suggests that PMTB2.1 control their iron requirements in response to iron availability by down-regulating the expression of iron proteins. Moreover, significantly high relative fold change (p ≤ 0.05) of fbpb gene is probably associated with the ability of P. multocida to directly use host iron complex such as hem, hemoglobin. In addition, the significant increase (p ≤ 0.05) in fbpb and yfea expressions also reflects the utilization of multiple iron systems in P. multocida strain PMTB2.1. The findings of this study are very much important as relative scarcity of free iron within hosts creates a major barrier to microbial growth inside host and utilization of outer-membrane proteins system in iron acquisition probably occurred at early stage of infection with P. multocida. In conclusion, the presence and utilization of multiple iron system in P. multocida strain PMTB2.1 revealed the importance of iron in the survival of P. multocida.

Keywords: iron-related genes, real-time PCR, gene expression profiling, fold changes

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Authors: Wisam H. Benamer, Ehab A. Elfallah, Mohamed A. Elshaari, Farag A. Elshaari

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A challenge for laboratories is to provide bacterial identification and antibiotic sensitivity results within a short time. Hence, advancement in the required technology is desirable to improve timing, accuracy and quality. Even with the current advances in methods used for both phenotypic and genotypic identification of bacteria the need is there to develop method(s) that enhance the outcome of bacteriology laboratories in accuracy and time. The hypothesis introduced here is based on the assumption that the chromosome of any bacteria contains unique sequences that can be used for its identification and typing. The outcome of a pilot study designed to test this hypothesis is reported in this manuscript. Methods: The complete chromosome sequences of several bacterial species were downloaded to use as search targets for unique sequences. Visual basic and SQL server (2014) were used to generate a complete set of 18-base long primers, a process started with reverse translation of randomly chosen 6 amino acids to limit the number of the generated primers. In addition, the software used to scan the downloaded chromosomes using the generated primers for similarities was designed, and the resulting hits were classified according to the number of similar chromosomal sequences, i.e., unique or otherwise. Results: All primers that had identical/similar sequences in the selected genome sequence(s) were classified according to the number of hits in the chromosomes search. Those that were identical to a single site on a single bacterial chromosome were referred to as unique. On the other hand, most generated primers sequences were identical to multiple sites on a single or multiple chromosomes. Following scanning, the generated primers were classified based on ability to differentiate between medically important bacterial and the initial results looks promising. Conclusion: A simple strategy that started by generating primers was introduced; the primers were used to screen bacterial genomes for match. Primer(s) that were uniquely identical to specific DNA sequence on a specific bacterial chromosome were selected. The identified unique sequence can be used in different molecular diagnostic techniques, possibly to identify bacteria. In addition, a single primer that can identify multiple sites in a single chromosome can be exploited for region or genome identification. Although genomes sequences draft of isolates of organism DNA enable high throughput primer design using alignment strategy, and this enhances diagnostic performance in comparison to traditional molecular assays. In this method the generated primers can be used to identify an organism before the draft sequence is completed. In addition, the generated primers can be used to build a bank for easy access of the primers that can be used to identify bacteria.

Keywords: bacteria chromosome, bacterial identification, sequence, primer generation

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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: Khabat Barkhordari, Ali Mohammad Arabzadeh

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Hepatitis delta virus (HDV) is a RNA virus that needs the function of hepatitis B virus (HBV) for its propagation and assembly. Infection by HDV can occur spontaneously with HBV infection and cause acute hepatitis or develop as secondary infection in HBV suffering patients. Based on genome sequence analysis, HDV has several genotypes which show broad geographic and diverse clinical features. The aim of current study is determine the prevalence of hepatitis delta virus genotype in patients with positive HBsAg in Kerman province of Iran. This cross-sectional study a total of 400 patients with HBV infection attending the clinic center of Besat from 2012 to 2014 were included. We carried out ELISA to detect anti-HDV antibodies. Those testing positive were analyzed further for HDV-RNA and for genotyping using restriction fragment length polymorphism (RFLP) and RT-nested PCR- sequencing. Among 400 patients in this study, 67 cases (16.75 %) were containing anti-HDV antibody which we found HDV RNA in just 7 (1.75%) serum samples. Analysis of these 7 positive HDV showed that all of them have genotype I. According to current study the HDV prevalence in Kerman is higher than the reported prevalence of 6.6% for Iran as a whole and clade 1 (genotype 1) is the predominant clade of HDV in Kerman.

Keywords: genotyping, hepatitis delta virus, molecular epidemiology, Kerman, Iran

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Authors: Aswini M. S.

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Tomato (Solanum lycopersicum L.) is one of the most significant vegetable crops in terms of its economic benefits. Both fresh and processed tomatoes are consumed. Tomatoes have a limited genetic base, which makes breeding extremely challenging. Plant breeding has become much simpler and more effective with genome editing tools of CRISPR and CRISPR-associated 9 protein (CRISPR/Cas9), which address the problems with traditional breeding, chemical/physical mutagenesis, and transgenics. With the use of CRISPR/Cas9, a number of tomato traits have been functionally distinguished and edited. These traits include plant architecture as well as flower characters (leaf, flower, male sterility, and parthenocarpy), fruit ripening, quality and nutrition (lycopene, carotenoid, GABA, TSS, and shelf-life), disease resistance (late blight, TYLCV, and powdery mildew), tolerance to abiotic stress (heat, drought, and salinity) and resistance to herbicides. This study explores the potential of CRISPR/Cas9 genome editing for enhancing yield in tomato plants. The study utilized the CRISPR/Cas9 genome editing technology to functionally edit various traits in tomatoes. The de novo domestication of elite features from wild cousins to cultivated tomatoes and vice versa has been demonstrated by the introgression of CRISPR/Cas9. The CycB (Lycopene beta someri) gene-mediated Cas9 editing increased the lycopene content in tomato. Also, Cas9-mediated editing of the AGL6 (Agamous-like 6) gene resulted in parthenocarpic fruit development under heat-stress conditions. The advent of CRISPR/Cas has rendered it possible to use digital resources for single guide RNA design and multiplexing, cloning (such as Golden Gate cloning, GoldenBraid, etc.), creating robust CRISPR/Cas constructs, and implementing effective transformation protocols like the Agrobacterium and DNA free protoplast method for Cas9-gRNAs ribonucleoproteins (RNPs) complex. Additionally, homologous recombination (HR)-based gene knock-in (HKI) via geminivirus replicon and base/prime editing (Target-AID technology) remains possible. Hence, CRISPR/Cas facilitates fast and efficient breeding in the improvement of tomatoes.

Keywords: CRISPR-Cas, biotic and abiotic stress, flower and fruit traits, genome editing, polygenic trait, tomato and trait introgression

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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: Asmuddin Natsir, A. Mujnisa, Syahriani Syahrir, Marhamah Nadir, Nurul Purnomo

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Bacteria, protozoa, Archaea, and fungi are the dominant microorganisms found in the rumen ecosystem that has an important role in converting feed ingredients into components that can be digested and utilized by the livestock host. This study was conducted to assess the diversity of rumen bacteria of bali cattle raised under traditional farming condition. Three adult bali cattle were used in this experiment. The rumen fluid samples from the three experimental animals were obtained by the Stomach Tube method before the morning feeding. The results of study indicated that the Illumina sequencing was successful in identifying 301,589 sequences, averaging 100,533 sequences, from three rumen fluid samples of three cattle. Furthermore, based on the SILVA taxonomic database, there were 19 kinds of phyla that had been successfully identified. Of the 19 phyla, there were only two dominant groups across the three samples, namely Bacteroidetes and Firmicutes, with an average percentage of 83.68% and 13.43%, respectively. Other groups such as Synergistetes, Spirochaetae, Planctomycetes can also be identified but in relatively small percentage. At the genus level, there were 157 sequences obtained from all three samples. Of this number, the most dominant group was Prevotella 1 with a percentage of 71.82% followed by 6.94% of Christencenellaceae R-7 group. Other groups such as Prevotellaceae UCG-001, Ruminococcaceae NK4A214 group, Sphaerochaeta, Ruminococcus 2, Rikenellaceae RC9 gut group, Quinella were also identified but with very low percentages. The sequencing results were able to detect the presence of 3.06% and 3.92% respectively for uncultured rumen bacterium and uncultured bacterium. In conclusion, the results of this experiment can provide an opportunity for a better understanding of the rumen bacterial diversity of the bali cattle raised under traditional farming condition and insight regarding the uncultured rumen bacterium and uncultured bacterium that need to be further explored.

Keywords: 16S rRNA sequencing, bali cattle, rumen microbial diversity, uncultured rumen bacterium

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Authors: Wei Tian, Jie Liang, Hammad Naveed

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Beta-barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They carry out diverse biological functions, including pore formation, membrane anchoring, enzyme activity, and bacterial virulence. In addition, beta-barrel membrane proteins increasingly serve as scaffolds for bacterial surface display and nanopore-based DNA sequencing. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank and computational methods can help to understand their biophysical principles. We have developed a novel computational method to predict the 3D structure of beta-barrel membrane proteins using evolutionary coupling (EC) constraints and a reduced state space. Combined with an empirical potential function, we can successfully predict strand register at > 80% accuracy for a set of 49 non-homologous proteins with known structures. This is a significant improvement from previous results using EC alone (44%) and using empirical potential function alone (73%). Our method is general and can be applied to genome-wide structural prediction.

Keywords: beta-barrel membrane proteins, structure prediction, evolutionary constraints, reduced state space

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Authors: Mary Kay Bacallao

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New research on the human genome has revealed secrets to the variation in skin pigmentation found in all human populations. Application of this research to multicultural education has a profound effect on students from all backgrounds. This paper identifies the four locations in the human genome that code for variation in skin pigmentation worldwide. The research makes this new knowledge accessible to students of all ages as they participate in an art project that brings these scientific multicultural concepts to life. Students participate in the application of breakthrough scientific principles through hands-on art activities where they simulate the work of the DNA coding to create their own skin tone using the colors expressed to varying degrees in every people group. As students create their own artwork handprint from the pallet of colors, they realize that each color on the pallet is essential to creating every tone of skin. This research project serves to bring people together and appreciate the variety and diversity in skin tones. As students explore the variations, they create pigmentation with the use of the eumelanins, which are the black-brown sources of pigmentation, and the pheomelanins, which are the yellow-reddish-brown sources of pigmentation. The research project dispels myths about skin tones that have divided people in the past. As a group project, this research leads to greater appreciation and understanding of the diverse family groups.

Keywords: diversity, multicultural, skin pigmentation, eumelanins, pheomelanins, handprint, artwork, science, genome, human

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Authors: Javed Mohammed

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Right now, bio-medical infrastructure lags well behind the curve. Our healthcare system is dispersed and disjointed; medical records are a bit of a mess; and we do not yet have the capacity to store and process the crazy amounts of data coming our way from widespread whole-genome sequencing. And then there are privacy issues. Despite these infrastructure challenges, some researchers are plunging into bio medical Big Data now, in hopes of extracting new and actionable knowledge. They are doing delving into molecular-level data to discover bio markers that help classify patients based on their response to existing treatments; and pushing their results out to physicians in novel and creative ways. Computer scientists and bio medical researchers are able to transform data into models and simulations that will enable scientists for the first time to gain a profound under-standing of the deepest biological functions. Solving biological problems may require High-Performance Computing HPC due either to the massive parallel computation required to solve a particular problem or to algorithmic complexity that may range from difficult to intractable. Many problems involve seemingly well-behaved polynomial time algorithms (such as all-to-all comparisons) but have massive computational requirements due to the large data sets that must be analyzed. High-throughput techniques for DNA sequencing and analysis of gene expression have led to exponential growth in the amount of publicly available genomic data. With the increased availability of genomic data traditional database approaches are no longer sufficient for rapidly performing life science queries involving the fusion of data types. Computing systems are now so powerful it is possible for researchers to consider modeling the folding of a protein or even the simulation of an entire human body. This research paper emphasizes the computational biology's growing need for high-performance computing and Big Data. It illustrates this article’s indispensability in meeting the scientific and engineering challenges of the twenty-first century, and how Protein Folding (the structure and function of proteins) and Phylogeny Reconstruction (evolutionary history of a group of genes) can use HPC that provides sufficient capability for evaluating or solving more limited but meaningful instances. This article also indicates solutions to optimization problems, and benefits Big Data and Computational Biology. The article illustrates the Current State-of-the-Art and Future-Generation Biology of HPC Computing with Big Data.

Keywords: high performance, big data, parallel computation, molecular data, computational biology

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Authors: Mohammad Vahed, Ana Sadeghitohidi, Majid Vahed, Hiroki Takahashi

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In the genomics field, the huge amounts of data have produced by the next-generation sequencers (NGS). Data volumes are very rapidly growing, as it is postulated that more than one billion bases will be produced per year in 2020. The growth rate of produced data is much faster than Moore's law in computer technology. This makes it more difficult to deal with genomics data, such as storing data, searching information, and finding the hidden information. It is required to develop the analysis platform for genomics big data. Cloud computing newly developed enables us to deal with big data more efficiently. Hadoop is one of the frameworks distributed computing and relies upon the core of a Big Data as a Service (BDaaS). Although many services have adopted this technology, e.g. amazon, there are a few applications in the biology field. Here, we propose a new algorithm to more efficiently deal with the genomics big data, e.g. sequencing data. Our algorithm consists of two parts: First is that BDaaS is applied for handling the data more efficiently. Second is that the hybrid method of MapReduce and Fuzzy logic is applied for data processing. This step can be parallelized in implementation. Our algorithm has great potential in computational analysis of genomics big data, e.g. de novo genome assembly and sequence similarity search. We will discuss our algorithm and its feasibility.

Keywords: big data, fuzzy logic, MapReduce, Hadoop, cloud computing

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Authors: Qiao Fan, Xiaobo Guo, Junxian Zhu, Xiaohu Ding, Ching-Yu Cheng, Tien-Yin Wong, Mingguang He, Heping Zhang, Xueqin Wang

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A systematic, simultaneous analysis of multiple phenotypes in genome-wide association studies (GWASs) draws a great attention to integrate the signals from single phenotypes with increased power. However, lacking an interpretable and efficient multivariate GWAS analysis impede the application of such approach. In this study, we propose to decompose the multivariate model into a series of simple univariate models. This transformation illuminates what exactly the individual trait contributes to the significant signals from the multivariate analyses. By employing our approach in the analysis of three myopia-related endophenotypes from the Singapore Malay Eye Study (SIMES), we identify novel candidate loci which were successfully validated in an independent Guangzhou Twin Eye Study (GTES).

Keywords: GWAS multivariate, multiple traits, myopia, association

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Authors: R. Hasterok, A. Betekhtin, N. Borowska, A. Braszewska-Zalewska, E. Breda, K. Chwialkowska, R. Gorkiewicz, D. Idziak, J. Kwasniewska, M. Kwasniewski, D. Siwinska, A. Wiszynska, E. Wolny

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In contrast to animals, the organisation of plant genomes at the cytomolecular level is still relatively poorly studied and understood. However, the Brachypodium genus in general and B. distachyon in particular represent exceptionally good model systems for such study. This is due not only to their highly desirable ‘model’ biological features, such as small nuclear genome, low chromosome number and complex phylogenetic relations, but also to the rapidly and continuously growing repertoire of experimental tools, such as large collections of accessions, WGS information, large insert (BAC) libraries of genomic DNA, etc. Advanced cytomolecular techniques, such as fluorescence in situ hybridisation (FISH) with evermore sophisticated probes, empowered by cutting-edge microscope and digital image acquisition and processing systems, offer unprecedented insight into chromatin organisation at various phases of the cell cycle. A good example is chromosome painting which uses pools of chromosome-specific BAC clones, and enables the tracking of individual chromosomes not only during cell division but also during interphase. This presentation outlines the present status of molecular cytogenetic analyses of plant genome structure, dynamics and evolution using B. distachyon and some of its relatives. The current projects focus on important scientific questions, such as: What mechanisms shape the karyotypes? Is the distribution of individual chromosomes within an interphase nucleus determined? Are there hot spots of structural rearrangement in Brachypodium chromosomes? Which epigenetic processes play a crucial role in B. distachyon embryo development and selective silencing of rRNA genes in Brachypodium allopolyploids? The authors acknowledge financial support from the Polish National Science Centre (grants no. 2012/04/A/NZ3/00572 and 2011/01/B/NZ3/00177)

Keywords: Brachypodium, B. distachyon, chromosome, FISH, molecular cytogenetics, nucleus, plant genome organisation

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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: Hamid Mustafa, Huson J. Heather, Kim Eiusoo, Adeela Ajmal, Tad S. Sonstegard

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The objective of this study was to evaluate the informativeness of Bovine high density SNPs genotyping in Sahiwal cattle population. This is a first attempt to assess the Bovine HD SNP genotyping array in any Pakistani indigenous cattle population. To evaluate these SNPs on genome wide scale, we considered 777,962 SNPs spanning the whole autosomal and X chromosomes in Sahiwal cattle population. Fifteen (15) non related gDNA samples were genotyped with the bovine HD infinium. Approximately 500,939 SNPs were found polymorphic (MAF > 0.05) in Sahiwal cattle population. The results of this study indicate potential application of Bovine High Density SNP genotyping in Pakistani indigenous cattle population. The information generated from this array can be applied in genetic prediction, characterization and genome wide association studies of Pakistani Sahiwal cattle population.

Keywords: Sahiwal cattle, polymorphic SNPs, genotyping, Pakistan

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Authors: Narin Salehiyan, Ramin Ghasemi Shayan

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The largest genus of plant viruses, the potyvirus, is responsible for significant crop losses. Potyviruses are aphid sent in a nonpersistent way, and some of them are likewise seed communicated. As significant microorganisms, potyviruses are substantially more examined than other plant infections having a place with different genera, and their review covers numerous parts of plant virology, like utilitarian portrayal of viral proteins, sub-atomic communication with hosts and vectors, structure, scientific classification, development, the study of disease transmission, and determination. Biotechnological utilizations of potyviruses are likewise being investigated. During this last ten years, significant advances have been made in the comprehension of the sub-atomic science of these infections and the elements of their different proteins. Potyvirus multiplication, movement, and transmission, as well as potyvirus/plant compatible interactions, including pathogenicity and symptom determinants, are updated following a general overview of the family Potyviridae and the potyviral proteins. it end the survey giving data on biotechnological uses of potyviruses.

Keywords: virology, poty, virus, genome, genetic

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Authors: M. Irfan Fareed, Muhammad Tahir, Alvina Gul Kazi

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Castor bean (Ricinus communis; family Euphorbiaceae) is cultivated for the production of oil and as an ornamental plant throughout tropical regions. Leaf samples from castor bean plants with leaf curl and vein thickening were collected from areas around Okara (Pakistan) in 2011. PCR amplification using diagnostic primers showed the presence of a begomovirus and subsequently the specific pair (BurNF 5’- CCATGGTTGTGGCAGTTGATTGACAGATAC-3’, BurNR 5’- CCATGGATTCACGCACAGGGGAACCC-3’) was used to amplify and clone the whole genome of the virus. The complete nucleotide sequence was determined to be 2,759 nt (accession No. HE985227). Alignments showed the highest levels of nucleotide sequence identity (98.8%) with Cotton leaf curl Burewala virus (CLCuBuV; accession No. JF416947) No. JF416947). The virus in castor beans lacks on intact C2 gene, as is typical of CLCuBuV in cotton. An amplification product of ca. 1.4 kb was obtained in PCR with primers for betasatellites and the complete nucleotide sequence of a clone was determined to be 1373 nt (HE985228). The sequence showed 96.3% nucleotide sequence identity to the recombinant Cotton leaf curl Multan betasatellite (CLCuMB; JF502389). This is the first report of CLCuBuV and its betasatellite infecting castor bean, showing this plant species as an alternate host of the virus. Already many alternate host have been reported from different alternate host like tobacco, tomato, hibiscus, okra, ageratum, Digera arvensis, habiscus, Papaya and now in Ricinus communis. So, it is suggested that these alternate hosts should be avoided to grow near cotton growing regions.

Keywords: Ricinus communis, begomovirus, betasatellite, agriculture

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Authors: Jafar Ahmadi, Zhohreh Asiaban, Sedigheh Fabriki Ourang

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Brassinosteroids (BRs) regulate cell elongation, vascular differentiation, senescence and stress responses. BRs signal through the BES1/BZR1 family of transcription factors, which regulate hundreds of target genes involved in this pathway. In this research a comprehensive genome-wide analysis was carried out in BES1/BZR1 gene family in Arabidopsis thaliana, Cucumis sativus, Vitis vinifera, Glycin max, and Brachypodium distachyon. Specifications of the desired sequences, dot plot and hydropathy plot were analyzed in the protein and genome sequences of five plant species. The maximum amino acid length was attributed to protein sequence Brdic3g with 374aa and the minimum amino acid length was attributed to protein sequence Gm7g with 163aa. The maximum Instability index was attributed to protein sequence AT1G19350 equal with 79.99 and the minimum Instability index was attributed to protein sequence Gm5g equal with 33.22. Aliphatic index of these protein sequences ranged from 47.82 to 78.79 in Arabidopsis thaliana, 49.91 to 57.50 in Vitis vinifera, 55.09 to 82.43 in Glycin max, 54.09 to 54.28 in Brachypodium distachyon 55.36 to 56.83 in Cucumis sativus. Overall, data obtained from our investigation contributes a better understanding of the complexity of the BES1/BZR1 gene family and provides the first step towards directing future experimental designs to perform systematic analysis of the functions of the BES1/BZR1 gene family.

Keywords: BES1/BZR1, brassinosteroids, phylogenetic analysis, transcription factor

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Authors: Muhammad Shahzad Iqbal, Zobia Sarwar, Salah-ud-Din

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Tomato spotted wilt virus (TSWV) belongs to the genus Tospoviruses (family Bunyaviridae). It is one of the most devastating pathogens of tomato (Solanum Lycopersicum) and heavily damages the crop yield each year around the globe. In this study, we retrieved 329 mature miRNA sequences from two microRNA databases (miRBase and miRSoldb) and checked the putative target sites in the downloaded-genome sequence of TSWV. A consensus of three miRNA target prediction tools (RNA22, miRanda and psRNATarget) was used to screen the false-positive microRNAs targeting sites in the TSWV genome. These tools calculated different target sites by calculating minimum free energy (mfe), site-complementarity, minimum folding energy and other microRNA-mRNA binding factors. R language was used to plot the predicted target-site data. All the genes having possible target sites for different miRNAs were screened by building a consensus table. Out of these 329 mature miRNAs predicted by three algorithms, only eight miRNAs met all the criteria/threshold specifications. MC-Fold and MC-Sym were used to predict three-dimensional structures of miRNAs and further analyzed in USCF chimera to visualize the structural and conformational changes before and after microRNA-mRNA interactions. The results of the current study show that the predicted eight miRNAs could further be evaluated by in vitro experiments to develop TSWV-resistant transgenic tomato plants in the future.

Keywords: tomato spotted wild virus (TSWV), Solanum lycopersicum, plant virus, miRNAs, microRNA target prediction, mRNA

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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

Abstract:

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

Procedia PDF Downloads 163