Search results for: Genome sequences

Authors: Sukrat Sinha, Abhay Kumar, Shanthy Sundaram

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The Leishmania homologue of activated C kinase (LACK) is known T cell epitope from soluble Leishmania antigens (SLA) that confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains. LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of visceral Leishmaniasis vaccine target.

Keywords: bioinformatics, genome assembly, leishmania activated protein kinase c (lack), next-generation sequencing

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

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

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

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Authors: Yina A. Cifuentes Triana, Andrés M. Pinzón Velásco, Marío E. Velásquez Lozano

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In this work the sequencing and genome characterization of a natural isolate of Saccharomyces cerevisiae yeast (strain 202-3), identified with potential for the production of second generation ethanol from sugarcane bagasse hydrolysates is presented. This strain was selected because its capability to consume xylose during the fermentation of sugarcane bagasse hydrolysates, taking into account that many strains of S. cerevisiae are incapable of processing this sugar. This advantage and other prominent positive aspects during fermentation profiles evaluated in bagasse hydrolysates made the strain 202-3 a candidate strain to improve the production of second-generation ethanol, which was proposed as a first step to study the strain at the genomic level. The molecular characterization was carried out by genome sequencing with the Illumina HiSeq 2000 platform paired end; the assembly was performed with different programs, finally choosing the assembler ABYSS with kmer 89. Gene prediction was developed with the approach of hidden Markov models with Augustus. The genes identified were scored based on similarity with public databases of nucleotide and protein. Records were organized from ontological functions at different hierarchical levels, which identified central metabolic functions and roles of the S. cerevisiae strain 202-3, highlighting the presence of four possible new proteins, two of them probably associated with the positive consumption of xylose.

Keywords: cellulosic ethanol, Saccharomyces cerevisiae, genome sequencing, xylose consumption

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Authors: Khin Thanda Win, Chunying Zhang, Sanghyeob Lee

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Mildew resistance locus o (Mlo), a plant-specific gene family with seven-transmembrane (TM), plays an important role in plant resistance to powdery mildew (PM). PM caused by Podosphaera xanthii is a widespread plant disease and probably represents the major fungal threat for many Cucurbits. The recent Cucurbita maxima genome sequence data provides an opportunity to identify and characterize the MLO gene family in this species. Total twenty genes (designated CmaMLO1 through CmaMLO20) have been identified by using an in silico cloning method with the MLO gene sequences of Cucumis sativus, Cucumis melo, Citrullus lanatus and Cucurbita pepo as probes. These CmaMLOs were evenly distributed on 15 chromosomes of 20 C. maxima chromosomes without any obvious clustering. Multiple sequence alignment showed that the common structural features of MLO gene family, such as TM domains, a calmodulin-binding domain and 30 important amino acid residues for MLO function, were well conserved. Phylogenetic analysis of the CmaMLO genes and other plant species reveals seven different clades (I through VII) and only clade IV is specific to monocots (rice, barley, and wheat). Phylogenetic and structural analyses provided preliminary evidence that five genes belonged to clade V could be the susceptibility genes which may play the importance role in PM resistance. This study is the first comprehensive report on MLO genes in C. maxima to our knowledge. These findings will facilitate the functional analysis of the MLOs related to PM susceptibility and are valuable resources for the development of disease resistance in pumpkin.

Keywords: Mildew resistance locus o (Mlo), powdery mildew, phylogenetic relationship, susceptibility genes

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Authors: Noshaba Dilbar, Aisha Tahir, Amer Jamil

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During the last decade, DNA barcoding proved to be an authentic tool for discovery and identification of plants. In the present study, DNA barcoding of two species, Fagonia arabica and Fagonia indica was done for differentiation by using matK region. matK gene is considered as a universal barcode because of its easy alignment and high discrimination ability. In this study, matK yielded 100% sequencing results. The sequences from both plants were aligned at clustal W and observed that there is no nucleotide variation and polymorphism among both sequences. This was further analysed by BLAST which showed the similar sequences from different plants belonging to same family but didn’t find sequence of both species. Considering this, the resulted sequence was submitted by the name of Fagonia arabica with accession number KM276890. In the end, we analysed the results from BOLD which gave us the final conclusion that both plants are same as their matK sequences are 100% identical. In literature, both Fagonia indica and Fagonia arabica names are used for this plant but there is no clear differentiation has been observed in these plants. Results evaluate that Fagonia indica and Fagonia arabica are the alternative names of same plant.

Keywords: DNA barcoding, Fagonia arabica, Fagonia indica, matK

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Authors: Neelma Ashraf

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Actinomycetes, particularly genus Streptomyces is of great importance due to their role in the discovery of new natural products, particularly antimicrobial secondary metabolites in the medicinal science and biotechnology industry. Different Streptomyces strains were isolated from Helianthus annuus plants and tested for antibacterial and antifungal activities. The most promising five strains were chosen for further investigation, and growth conditions for antibiotic synthesis were optimised. The supernatants were extracted in different solvents, and the extracted products were analyzed using liquid chromatography-mass spectrometry (LC-MS) and biological testing. From one of the potent strains Streptomyces globusus sp. BR123, a compound lavendamycin was identified using these analytical techniques. In addition, this potent strain also produces a strong antifungal polyene compound with a quasimolecular ion of 2072. Streptomyces sp. BR123 was genome sequenced because of its promising antimicrobial potential in order to identify the gene cluster responsible for analyzed compound “lavendamycin”. The genome analysis yielded candidate genes responsible for the production of this potent compound. The genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 with a GC content of 72.63% and 8103 protein coding genes was attained. Many antimicrobial, antiparasitic, and anticancerous compounds were detected through multiple biosynthetic gene clusters predicted by in-Silico analysis. Though, the novelty of metabolites was determined through the insignificant resemblance with known biosynthetic gene clusters. The current study gives insight into the bioactive potential of Streptomyces sp. isolate BR123 with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study revealed the connection of isolate BR123 with other Streptomyces strains, which could expand the knowledge of this genus and the mechanism involved in the discovery of new antimicrobial metabolites.

Keywords: streptomyces, secondary metabolites, genome, biosynthetic gene clusters, high performance liquid chromatography, mass spectrometry

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Authors: Afsaneh Mohammadnejad, Marianne Nygaard, Jan Baumbach, Shuxia Li, Weilong Li, Jesper Lund, Jacob v. B. Hjelmborg, Lene Christensen, Qihua Tan

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Cognitive impairment in the elderly is a key issue affecting the quality of life. Despite a strong genetic background in cognition, only a limited number of single nucleotide polymorphisms (SNPs) have been found. These explain a small proportion of the genetic component of cognitive function, thus leaving a large proportion unaccounted for. We hypothesize that one reason for this missing heritability is the misspecified modeling in data analysis concerning phenotype distribution as well as the relationship between SNP dosage and the phenotype of interest. In an attempt to overcome these issues, we introduced a model-free method based on the generalized correlation coefficient (GCC) in a genome-wide association study (GWAS) of cognitive function in twin samples and compared its performance with two popular linear regression models. The GCC-based GWAS identified two genome-wide significant (P-value < 5e-8) SNPs; rs2904650 near ZDHHC2 on chromosome 8 and rs111256489 near CD6 on chromosome 11. The kinship model also detected two genome-wide significant SNPs, rs112169253 on chromosome 4 and rs17417920 on chromosome 7, whereas no genome-wide significant SNPs were found by the linear mixed model (LME). Compared to the linear models, more meaningful biological pathways like GABA receptor activation, ion channel transport, neuroactive ligand-receptor interaction, and the renin-angiotensin system were found to be enriched by SNPs from GCC. The GCC model outperformed the linear regression models by identifying more genome-wide significant genetic variants and more meaningful biological pathways related to cognitive function. Moreover, GCC-based GWAS was robust in handling genetically related twin samples, which is an important feature in handling genetic confounding in association studies.

Keywords: cognition, generalized correlation coefficient, GWAS, twins

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Authors: Surabhi Maiti, Prajakta Tamhankar, Prachi Uttam Mehta

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Since the accomplishment of the Human Genome Project, there has been an unparalled escalation in the sequencing of genomic data. This project has been the first major vault in the field of medical research, especially in genomics. This project won accolades by using a concept called Bigdata which was earlier, extensively used to gain value for business. Bigdata makes use of data sets which are generally in the form of files of size terabytes, petabytes, or exabytes and these data sets were traditionally used and managed using excel sheets and RDBMS. The voluminous data made the process tedious and time consuming and hence a stronger framework called Hadoop was introduced in the field of genetic sciences to make data processing faster and efficient. This paper focuses on using SPARK which is gaining momentum with the advancement of BigData technologies. Cloud Storage is an effective medium for storage of large data sets which is generated from the genetic research and the resultant sets produced from SPARK analysis.

Keywords: human genome project, Bigdata, genomic data, SPARK, cloud storage, Hadoop

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Authors: Motonobu Hattori

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In neural networks, when new patters are learned by a network, they radically interfere with previously stored patterns. This drawback is called catastrophic forgetting. We have already proposed a biologically inspired dual-network memory model which can much reduce this forgetting for static patterns. In this model, information is first stored in the hippocampal network, and thereafter, it is transferred to the neocortical network using pseudo patterns. Because, temporal sequence learning is more important than static pattern learning in the real world, in this study, we improve our conventional dual-network memory model so that it can deal with temporal sequences without catastrophic forgetting. The computer simulation results show the effectiveness of the proposed dual-network memory model.

Keywords: catastrophic forgetting, dual-network, temporal sequences, hippocampal

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Authors: Maxime Merheb, Rachel Matar

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Retrieving ancient DNA sequences which in return permit the entire genome sequencing from fossils have extraordinarily improved in recent years, thanks to sequencing technology and other methodological advances. In any case, the quest to search for ancient DNA is still obstructed by the damage inflicted on DNA which accumulates after the death of a living organism. We can characterize this damage into three main categories: (i) Physical abnormalities such as strand breaks which lead to the presence of short DNA fragments. (ii) Modified bases (mainly cytosine deamination) which cause errors in the sequence due to an incorporation of a false nucleotide during DNA amplification. (iii) DNA modifications referred to as blocking lesions, will halt the PCR extension which in return will also affect the amplification and sequencing process. We can clearly see that the issues arising from breakage and coding errors were significantly decreased in recent years. Fast sequencing of short DNA fragments was empowered by platforms for high-throughput sequencing, most of the coding errors were uncovered to be the consequences of cytosine deamination which can be easily removed from the DNA using enzymatic treatment. The methodology to repair DNA sequences is still in development, it can be basically explained by the process of reintroducing cytosine rather than uracil. This technique is thus restricted to amplified DNA molecules. To eliminate any type of damage (particularly those that block PCR) is a process still pending the complete repair methodologies; DNA detection right after extraction is highly needed. Before using any resources into extensive, unreasonable and uncertain repair techniques, it is vital to distinguish between two possible hypotheses; (i) DNA is none existent to be amplified to begin with therefore completely un-repairable, (ii) the DNA is refractory to PCR and it is worth to be repaired and amplified. Hence, it is extremely important to develop a non-enzymatic technique to detect the most degraded DNA.

Keywords: ancient DNA, DNA barcodong, enzymatic repair, PCR

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Authors: Borys Stegniy, Anton Gerilovych, Oleksii Solodiankin, Vitaliy Bolotin, Anton Stegniy, Denys Muzyka, Claudio Afonso

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New APMV was isolated from white fronted goose in Ukraine. This isolate was tested serologically using monoclonal antibodies in haemagglutination-inhibition tests against APMV1-9. As the results obtained isolate showed cross reactions with APMV7. Following investigations were provided for the full genome sequencing using random primers and cloning into pCRII-TOPO. Analysis of 100 transformed colonies of E.coli using traditional sequencing gave us possibilities to find only 3 regions, which could identify by BLAST. The first region with the length of 367 bp had 70 % nucleotide sequence identity to the APMV 12 isolate Wigeon/Italy/3920_1/2005 at genome position 2419-2784. Next region (344 bp) had 66 % identity to the same APMV 12 isolate at position 4760-5103. The last region (365 bp) showed 71 % identity to Newcastle disease virus strain M4 at position 12569-12928.

Keywords: APMV, Newcastle disease virus, Ukraine, full genome sequencing

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Authors: Umairah Natasya Binti Mohd Omeershffudin, Suresh Kumar

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Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. Particular concern is the global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae. Characterization of antibiotic resistance determinants at the genomic level plays a critical role in understanding, and potentially controlling, the spread of multidrug-resistant (MDR) pathogens. In this study, drug-resistant Klebsiella pneumoniae strain 825795-1 was investigated with extensive computational approaches aimed at identifying novel drug targets among hypothetical proteins. We have analyzed 1099 hypothetical proteins available in genome. We have used in-silico genome subtraction methodology to design potential and pathogen-specific drug targets against Klebsiella pneumoniae. We employed bioinformatics tools to subtract the strain-specific paralogous and host-specific homologous sequences from the bacterial proteome. The sorted 645 proteins were further refined to identify the essential genes in the pathogenic bacterium using the database of essential genes (DEG). We found 135 unique essential proteins in the target proteome that could be utilized as novel targets to design newer drugs. Further, we identified 49 cytoplasmic protein as potential drug targets through sub-cellular localization prediction. Further, we investigated these proteins in the DrugBank databases, and 11 of the unique essential proteins showed druggability according to the FDA approved drug bank databases with diverse broad-spectrum property. The results of this study will facilitate discovery of new drugs against Klebsiella pneumoniae.

Keywords: pneumonia, drug target, hypothetical protein, subtractive genomics

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Authors: Andrey V. Khromov, Antonida V. Makhotenko, Ekaterina A. Snigir, Svetlana S. Makarova, Natalia O. Kalinina, Valentin V. Makarov, Mikhail E. Taliansky

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Due to its simplicity, CRISPR/Cas9 has become widely used and capable of inducing mutations in the genes of organisms of various kingdoms. The aim of this work was to develop applications for the efficient modification of DNA coding sequences of phytoene desaturase (PDS), coilin and vacuolar invertase (Solanum tuberosum) genes, and to develop a new nanoparticles carrier efficient technology to deliver the CRISPR/Cas9 system for editing the plant genome. For each of the genes - coilin, PDS and vacuolar invertase, five single RNA guide (sgRNAs) were synthesized. To determine the most suitable nanoplatform, two types of NP platforms were used: magnetic NPs (MNPS) and gold NPs (AuNPs). To test the penetration efficiency, they were functionalized with fluorescent agents - BSA * FITS and GFP, as well as labeled Cy3 small-sized RNA. To measure the efficiency, a fluorescence and confocal microscopy were used. It was shown that the best of these options were AuNP - both in the case of proteins and in the case of RNA. The next step was to check the possibility of delivering components of the CRISPR/Cas9 system to plant cells for editing target genes. AuNPs were functionalized with a ribonucleoprotein complex consisting of Cas9 and corresponding to target genes sgRNAs, and they were biolistically bombarded to axillary buds and apical meristems of potato plants. After the treatment by the best NP carrier, potato meristems were grown to adult plants. DNA isolated from this plants was sent to a preliminary fragment of the analysis to screen out the non-transformed samples, and then to the NGS. The present work was carried out with the financial support from the Russian Science Foundation (grant No. 16-16-04019).

Keywords: biobombardment, coilin, CRISPR/Cas9, nanoparticles, NPs, PDS, sgRNA, vacuolar invertase

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Authors: Muhammad A. Khan, Waseem Shahzad

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Analysis of protein sequences is carried out for the purpose to discover their structural and ancestry relationship. Sequence similarity determines similar protein structures, similar function, and homology detection. Biological sequences composed of amino acid residues or nucleotides provide significant information through sequence alignment. In this paper, we present a new similarity/dissimilarity measure to sequence alignment based on the primary structure of a protein. The approach finds the distance between the two given sequences using the novel sequence alignment algorithm and a mathematical model. The algorithm runs at a time complexity of O(n²). A distance matrix is generated to construct a phylogenetic tree of different species. The new similarity/dissimilarity measure outperforms other existing methods.

Keywords: alignment, distance, homology, mathematical model, phylogenetic tree

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

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

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

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Authors: Tulin Coskun

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We investigate the approximation of analytic functions of several variables in polydisc by the sequences of linear k-positive operators in Gadjiev sence. The approximation of analytic functions of complex variable by linear k-positive operators was tackled, and k-positive operators and formulated theorems of Korovkin's type for these operators in the space of analytic functions on the unit disc were introduced in the past. Recently, very general results on convergence of the sequences of linear k-positive operators on a simply connected bounded domain within the space of analytic functions were proved. In this presentation, we extend some of these results to the approximation of analytic functions of several complex variables by sequences of linear k-positive operators.

Keywords: analytic functions, approximation of analytic functions, Linear k-positive operators, Korovkin type theorems

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Authors: Ghulam Muhammad Ali

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Unveiling of genes involved in drought and root architecture using transcriptomic analyses remained fragmented for further improvement of wheat through genome editing. The purpose of this research endeavor was to unveil the variations in different genes implicated in drought tolerance and root architecture in wheat through RNA-seq data analysis. In this study seedlings of 8 days old, 6 cultivars of wheat namely, Batis, Blue Silver, Local White, UZ888, Chakwal 50 and Synthetic wheat S22 were subjected to transcriptomic analysis for root and shoot genes. Total of 12 RNA samples was sequenced by Illumina. Using updated wheat transcripts from Ensembl and IWGC references with 54,175 gene models, we found that 49,621 out of 54,175 (91.5%) genes are expressed at an RPKM of 0.1 or more (in at least 1 sample). The number of genes expressed was higher in Local White than Batis. Differentially expressed genes (DEG) were higher in Chakwal 50. Expression-based clustering indicated conserved function of DRO1and RPK1 between Arabidopsis and wheat. Dendrogram showed that Local White is sister to Chakwal 50 while Batis is closely related to Blue Silver. This study flaunts transcriptomic sequence variations in different cultivars that showed mutations in genes associated with drought that may directly contribute to drought tolerance. DRO1 and RPK1 genes were fetched/isolated for genome editing. These genes are being edited in wheat through CRISPR-Cas9 for yield enhancement.

Keywords: transcriptomic, wheat, genome editing, drought, CRISPR-Cas9, yield enhancement

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Authors: Bin Liu

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Prokaryotic promoter, consisted of two short DNA sequences located at in -35 and -10 positions, is responsible for controlling the initiation and expression of gene expression. Different types of promoters have different functions, and their consensus sequences are similar. In addition, their consensus sequences may be different for the same type of promoter, which poses difficulties for promoter identification. Unfortunately, all existing computational methods treat promoter identification as a binary classification task and can only identify whether a query sequence belongs to a specific promoter type. It is desired to develop computational methods for effectively identifying promoters and their types. Here, a two-layer predictor is proposed to try to deal with the problem. The first layer is designed to predict whether a given sequence is a promoter and the second layer predicts the type of promoter that is judged as a promoter. Meanwhile, we also analyze the importance of feature and sequence conversation in two aspects: promoter identification and promoter type identification. To the best knowledge of ours, it is the first computational predictor to detect promoters and their types.

Keywords: promoter, promoter type, random forest, sequence information

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Authors: Vinod Kumar Gupta, Narendrakumar M. Chaudhari, Suchismitha Iskepalli, Chitra Dutta

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Background-The community composition of the human microbiome is known to vary at distinct anatomical niches. But little is known about the nature of variations if any, at the genome/sub-genome levels of a specific microbial community across different niches. The present report aims to explore, as a case study, the variations in gene repertoire of 28 Prevotella reference draft genomes derived from different body-sites of human, as reported earlier by the Human Microbiome Consortium. Results-The analysis reveals the exclusive presence of 11798, 3673, 3348 and 934 gene families and exclusive absence of 17, 221, 115 and 645 gene families in Prevotella genomes derived from the human oral cavity, gastro-intestinal tracts (GIT), urogenital tract (UGT) and skin, respectively. The pan-genome for Prevotella remains “open”. Distribution of various functional COG categories differs appreciably among the habitat-specific genes, within Prevotella pan-genome and between the GIT-derived Bacteroides and Prevotella. The skin and GIT isolates of Prevotella are enriched in singletons involved in Signal transduction mechanisms, while the UGT and oral isolates show higher representation of the Defense mechanisms category. No niche-specific variations could be observed in the distribution of KEGG pathways. Conclusion-Prevotella may have developed distinct genetic strategies for adaptation to different anatomical habitats through selective, niche-specific acquisition and elimination of suitable gene-families. In addition, individual microorganisms tend to develop their own distinctive adaptive stratagems through large repertoires of singletons. Such in situ, habitat-driven refurbishment of the genetic makeup can impart substantial intra-lineage genome diversity within the microbes without perturbing their general taxonomic heritage.

Keywords: body niche adaptation, human microbiome, pangenome, Prevotella

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Authors: Adel M. Zakri, Mohammed A. Al-Saleh, Judith. K. Brown, Ali M. Idris

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Betasatellites are small circular single stranded DNA molecules found associated with begomoviruses on field symptomatic plants. Their genome size is about half that of the helper begomovirus, ranging between 1.3 and 1.4 kb. The helper begomoviruses are usually members of the family Geminiviridae. Okra leaves showing vein thickening were collected from okra plants growing in Jazan, Saudi Arabia. Total DNA was extracted from leaves and used as a template to amplify circular DNA using rolling circle amplification (RCA) technology. Products were digested with PstI to linearize the helper viral genome(s), and associated DNA satellite(s), yielding a 2.8kbp and 1.4kbp fragment, respectively. The linearized fragments were cloned into the pGEM-5Zf (+) vector and subjected to DNA sequencing. The 2.8 kb fragment was identified as Cotton leaf curl Gezira virus genome, at 2780bp, an isolate closely related to strains reported previously from Saudi Arabia. A clone obtained from the 1.4 kb fragments he 1.4kb was blasted to GeneBank database found to be a betasatellite. The genome of betasatellite was 1357-bp in size. It was found to be a recombinant containing one fragment (877-bp) that shared 91% nt identity with Cotton leaf curl Gezira betasatellite [KM279620], and a smaller fragment [133--bp) that shared 86% nt identity with Tomato leaf curl Sudan virus [JX483708]. This satellite is thus a recombinant between a malvaceous-infecting satellite and a solanaceous-infecting begomovirus.

Keywords: begomovirus, betasatellites, cotton leaf curl Gezira virus, okra plants

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Authors: Abu S. Mustafa, Mohammad W. Khan, Faraz Shaheed Khan, Nazima Habibi

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Brucellosis is a zoonotic disease of worldwide prevalence. There are at least four species and several strains of Brucella that cause human disease. Brucella genomes have very limited variation across strains, which hinder strain identification using classical molecular techniques, including PCR and 16 S rDNA sequencing. The aim of this study was to perform whole genome sequencing of clinical isolates of Brucella and perform bioinformatics and comparative genomics analyses to determine the existence of genetic differences across the isolates of a single Brucella species and strain. The draft sequence data were generated from 15 clinical isolates of Brucella melitensis (biovar 2 strain 63/9) using MiSeq next generation sequencing platform. The generated reads were used for further assembly and analysis. All the analysis was performed using Bioinformatics work station (8 core i7 processor, 8GB RAM with Bio-Linux operating system). FastQC was used to determine the quality of reads and low quality reads were trimmed or eliminated using Fastx_trimmer. Assembly was done by using Velvet and ABySS softwares. The ordering of assembled contigs was performed by Mauve. An online server RAST was employed to annotate the contigs assembly. Annotated genomes were compared using Mauve and ACT tools. The QC score for DNA sequence data, generated by MiSeq, was higher than 30 for 80% of reads with more than 100x coverage, which suggested that data could be utilized for further analysis. However when analyzed by FastQC, quality of four reads was not good enough for creating a complete genome draft so remaining 11 samples were used for further analysis. The comparative genome analyses showed that despite sharing same gene sets, single nucleotide polymorphisms and insertions/deletions existed across different genomes, which provided a variable extent of diversity to these bacteria. In conclusion, the next generation sequencing, bioinformatics, and comparative genome analysis can be utilized to find variations (point mutations, insertions and deletions) across different genomes of Brucella within a single strain. This information could be useful in surveillance and epidemiological studies supported by Kuwait University Research Sector grants MI04/15 and SRUL02/13.

Keywords: brucella, bioinformatics, comparative genomics, whole genome sequencing

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Authors: Alsu F. Saifitdinova, Alexey S. Komissarov, Svetlana A. Galkina, Elena I. Koshel, Maria M. Kulak, Stephen J. O'Brien, Elena R. Gaginskaya

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The mystery of sex determination is one of the most ancient and still not solved until the end so far. In many species, sex determination is genetic and often accompanied by the presence of dimorphic sex chromosomes in the karyotype. Genomic sequencing gave the information about the gene content of sex chromosomes which allowed to reveal their origin from ordinary autosomes and to trace their evolutionary history. Female-specific W chromosome in birds as well as mammalian male-specific Y chromosome is characterized by the degeneration of gene content and the accumulation of repetitive DNA. Tandem repeats complicate the analysis of genomic data. Despite the best efforts chicken W chromosome assembly includes only 1.2 Mb from expected 55 Mb. Supplementing the information on the sex chromosome composition not only helps to complete the assembly of genomes but also moves us in the direction of understanding of the sex-determination systems evolution. A whole-genome survey to the assembly Gallus_gallus WASHUC 2.60 was applied for repeats search in assembled genome and performed search and assembly of high copy number repeats in unassembled reads of SRR867748 short reads datasets. For cytogenetic analysis conventional methods of fluorescent in situ hybridization was used for previously cloned W specific satellites and specifically designed directly labeled synthetic oligonucleotide DNA probe was used for bioinformatically identified repetitive sequence. Hybridization was performed with mitotic chicken chromosomes and manually isolated giant meiotic lampbrush chromosomes from growing oocytes. A novel chicken W specific satellite (GGAAA)n which is not co-localizes with any previously described classes of W specific repeats was identified and mapped with high resolution. In the composition of autosomes this repeat units was found as a part of upstream regions of gonad specific protein coding sequences. These findings may contribute to the understanding of the role of tandem repeats in sex specific differentiation regulation in birds and sex chromosome evolution. This work was supported by the postdoctoral fellowships from St. Petersburg State University (#1.50.1623.2013 and #1.50.1043.2014), the grant for Leading Scientific Schools (#3553.2014.4) and the grant from Russian foundation for basic researches (#15-04-05684). The equipment and software of Research Resource Center “Chromas” and Theodosius Dobzhansky Center for Genome Bioinformatics of Saint Petersburg State University were used.

Keywords: birds, lampbrush chromosomes, sex chromosomes, tandem repeats

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Authors: Fatimazahrae Elbakri, Azeddine Ibrahimi, Meryem Lemrani, Dris Belghyti

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Leishmaniasis represents a major public health problem because of the number of cases recorded each year and the wide distribution of the disease. It is a parasitic disease of flagellated protozoa transmitted by the bite of certain species of sandfly, causing a spectrum of clinical pathology in humans ranging from disfiguring skin lesions to fatal visceral leishmaniasis. Cutaneous leishmaniasis due to Leishmania major is a polymorphic disease; in fact, the infection can be asymptomatic, localized, or disseminated. The objective of this work is to determine the genomic diversity that contributes to clinical variability by trying to identify the variation in chromosome number and to extract SNPs and SNPs and InDels; it is based on four sequences (WGS) of Leishmania major available on NCBI in Fastq form, from three countries: Tunisia, Algeria, and Israel, the analysis is set up from a pipeline to facilitate the discovery of genetic diversity, in particular SNP and chromosomal somy.

Keywords: Leshmania major, cutaneous Leishmania, NGS, genomic, somy, variant calling

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Authors: Saed Khawaldeh, Mohamed Elsharnouby, Alaa Eddin Alchalabi, Usama Pervaiz, Tajwar Aleef, Vu Hoang Minh

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Taxonomic classification has a wide-range of applications such as finding out more about the evolutionary history of organisms that can be done by making a comparison between species living now and species that lived in the past. This comparison can be made using different kinds of extracted species’ data which include DNA sequences. Compared to the estimated number of the organisms that nature harbours, humanity does not have a thorough comprehension of which specific species they all belong to, in spite of the significant development of science and scientific knowledge over many years. One of the methods that can be applied to extract information out of the study of organisms in this regard is to use the DNA sequence of a living organism as a marker, thus making it available to classify it into a taxonomy. The classification of living organisms can be done in many machine learning techniques including Neural Networks (NNs). In this study, DNA sequences classification is performed using Convolutional Neural Networks (CNNs) which is a special type of NNs.

Keywords: deep networks, convolutional neural networks, taxonomic classification, DNA sequences classification

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Authors: Anna Solovyeva, Ivan Levakin, Nickolai Galaktionov, Olga Podgornaya

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Animals that reproduce asexually were thought to have the same genotypes within generations for a long time. However, some refuting examples were found, and mobile genetic elements (MGEs) or transposons are considered to be the most probable source of genetic instability. Dispersed nature and the ability to change their genomic localization enables MGEs to be efficient mutators. Hence the study of MGEs genomic impact requires an appropriate object which comprehends both representative amounts of various MGEs and options to evaluate the genomic influence of MGEs. Animals that reproduce asexually seem to be a decent model to study MGEs impact in genomic variability. We found a small marine trematode Himasthla elongata (Himasthlidae) to be a good model for such investigation as it has a small genome size, diverse MGEs and parthenogenetic stages in the lifecycle. In the current work, clonal diversity of cercaria was traced with an AFLP (Amplified fragment length polymorphism) method, diverse zones from electrophoretic patterns were cloned, and the nature of the fragments explored. Polymorphic patterns of individual cercariae AFLP-based fingerprints are enriched with retrotransposons of different families. The bulk of those sequences are represented by open reading frames of non-Long Terminal Repeats containing elements(non-LTR) yet Long-Terminal Repeats containing elements (LTR), to a lesser extent in variable figments of AFLP array. The CR1 elements expose both in polymorphic and conservative patterns are remarkably more frequent than the other non-LTR retrotransposons. This data was confirmed with shotgun sequencing-based on Illumina HiSeq 2500 platform. Individual cercaria of the same clone (i.e., originated from a single miracidium and inhabiting one host) has a various distribution of MGE families detected in sequenced AFLP patterns. The most numerous are CR1 and RTE-Bov retrotransposons, typical for trematode genomes. Also, we identified LTR-retrotransposons of Pao and Gypsy families among DNA transposons of CMC-EnSpm, Tc1/Mariner, MuLE-MuDR and Merlin families. We detected many of them in H. elongata transcriptome. Such uneven MGEs distribution in AFLP sequences’ sets reflects the different patterns of transposons spreading in cercarial genomes as transposons affect the genome in many ways (ectopic recombination, gene structure interruption, epigenetic silencing). It is considered that they play a key role in the origins of trematode clonal polymorphism. The authors greatly appreciate the help received at the Kartesh White Sea Biological Station of the Russian Academy of Sciences Zoological Institute. This work is funded with RSF 19-74-20102 and RFBR 17-04-02161 grants and the research program of the Zoological Institute of the Russian Academy of Sciences (project number AAAA-A19-119020690109-2).

Keywords: AFLP, clonal polymorphism, Himasthla elongata, mobile genetic elements, NGS

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Authors: B. M. Md-Zain, N. A. Rahman, M. A. B. Abdul-Latiff, W. M. R. Idris

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We conducted molecular research to portray phylogenetic relationships of Malaysian primates particularly in the genus of Macaca. We have sequenced cytochrome C oxidase subunit I (COI) of mitochondrial DNA of several individuals from M. fascicularis and M. arctoides. PCR amplifications were performed and COI DNA sequences were aligned using ClustalW. Phylogenetic trees were constructed using distance analyses by employing neighbor-joining algorithm (NJ). We managed to sequence 700 bp of COI DNA sequences. The tree topology showed that M. fascicularis did not clump based on phyleogeography division in Peninsular Malaysia. Individuals from Negeri Sembilan merged together with samples from Perak and Penang into one clade. In addition, phylogenetic analyses indicated that M. arctoides was classified into sinica group instead of fascicularis group supported by genetic distance data. COI gene is an effective locus to clarify phylogenetic position of M. arctoides but not in discriminating M. fascicularis population in Peninsular Malaysia.

Keywords: cercopithecine, long-tailed macaque, Macaca fascicularis, Macaca arctoides

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Authors: Hana Barak, Alex Sivan, Ariel Kushmaro

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

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

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Authors: Judith Mogouong, Philippe Constant, Robert Lavallee, Claude Guertin

Abstract:

The emerald ash borer (EAB) is an exotic wood borer insect native from China, which is associated with important environmental and economic damages in North America. Beetles are known to be vectors of microbial communities related to their adaptive capacities. It is now established that environmental stress factors may induce physiological events on the host trees, such as phytochemical changes. Consequently, that may affect the establishment comportment of herbivorous insect. Considering the number of insects collected on ash trees (insects’ density) as an abiotic factor related to stress damage, the aim of our study was to explore the dynamic of EAB gut microbial community genome (microbiome) when facing that factor and to monitor its diversity. Insects were trapped using specific green Lindgren© traps. A gradient of the captured insect population along the St. Lawrence River was used to create three levels of insects’ density (low, intermediate, and high). After dissection, total DNA extracted from insect guts of each level has been sent for amplicon sequencing of bacterial 16S rRNA gene and fungal ITS2 region. The composition of microbial communities among sample appeared largely diversified with the Simpson index significantly different across the three levels of density for bacteria. Add to that; bacteria were represented by seven phyla and twelve classes, whereas fungi were represented by two phyla and seven known classes. Using principal coordinate analysis (PCoA) based on Bray Curtis distances of 16S rRNA sequences, we observed a significant variation between the structure of the bacterial communities depending on insects’ density. Moreover, the analysis showed significant correlations between some bacterial taxa and the three classes of insects’ density. This study is the first to present a complete overview of the bacterial and fungal communities associated with the gut of EAB base on culture-independent methods, and to correlate those communities with a potential stress factor of the host trees.

Keywords: gut microbiome, DNA, 16S rRNA sequences, emerald ash borer

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Authors: Agostinho Antunes

Abstract:

The completion of the human genome sequencing in 2003 opened a new perspective into the importance of whole genome sequencing projects, and currently multiple species are having their genomes completed sequenced, from simple organisms, such as bacteria, to more complex taxa, such as mammals. This voluminous sequencing data generated across multiple organisms provides also the framework to better understand the genetic makeup of such species and related ones, allowing to explore the genetic changes underlining the evolution of diverse phenotypic traits. Here, recent results from our group retrieved from comparative evolutionary genomic analyses of varied species will be considered to exemplify how gene novelty and gene enhancement by positive selection might have been determinant in the success of adaptive radiations into diverse habitats and lifestyles.

Keywords: adaptation, animals, evolution, genomics

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Authors: Maryam Saeidi, Ehsan Motamedian, Seyed Abbas Shojaosadati

Abstract:

Zymomonas mobilis is known as an example of the uncoupled growth phenomenon. This microorganism also has a unique metabolism that degrades glucose by the Entner–Doudoroff (ED) pathway. In this paper, a genome-scale metabolic model including 434 genes, 757 reactions and 691 metabolites was reconstructed to simulate uncoupled growth and study its effect on flux distribution in the central metabolism. The model properly predicted that ATPase was activated in experimental growth yields of Z. mobilis. Flux distribution obtained from model indicates that the major carbon flux passed through ED pathway that resulted in the production of ethanol. Small amounts of carbon source were entered into pentose phosphate pathway and TCA cycle to produce biomass precursors. Predicted flux distribution was in good agreement with experimental data. The model results also indicated that Z. mobilis metabolism is able to produce biomass with maximum growth yield of 123.7 g (mol glucose)-1 if ATP synthase is coupled with growth and produces 82 mmol ATP gDCW-1h-1. Coupling the growth and energy reduced ethanol secretion and changed the flux distribution to produce biomass precursors.

Keywords: genome-scale metabolic model, Zymomonas mobilis, uncoupled growth, flux distribution, ATP dissipation

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