Search results for: genome analysis

Authors: Abiodun Olayinka, Daniel Dzidzienyo, Pangirayi Tongoona, Samuel Offei, Edwige Gaby Nkouaya Mbanjo, Chiedozie Egesi, Ismail Yusuf Rabbi

Abstract:

Cassava (Manihot esculenta Crantz) is a major source of starch for various industrial applications. However, the traditional cultivation and harvesting methods of cassava are labour-intensive and inefficient, limiting the supply of fresh cassava roots for industrial starch production. To achieve improved productivity and quality of fresh cassava roots through mechanized cultivation, cassava cultivars with compact plant architecture and moderate plant height are needed. Plant architecture-related traits, such as plant height, harvest index, stem diameter, branching angle, and lodging tolerance, are critical for crop productivity and suitability for mechanized cultivation. However, the genetics of cassava plant architecture remain poorly understood. This study aimed to identify the genetic bases of the relationships between plant architecture traits and productivity-related traits, particularly starch content. A panel of 453 clones developed at the International Institute of Tropical Agriculture, Nigeria, was genotyped and phenotyped for 18 plant architecture and productivity-related traits at four locations in Nigeria. A genome-wide association study (GWAS) was conducted using the phenotypic data from a panel of 453 clones and 61,238 high-quality Diversity Arrays Technology sequencing (DArTseq) derived Single Nucleotide Polymorphism (SNP) markers that are evenly distributed across the cassava genome. Five significant associations between ten SNPs and three plant architecture component traits were identified through GWAS. We found five SNPs on chromosomes 6 and 16 that were significantly associated with shoot weight, harvest index, and total yield through genome-wide association mapping. We also discovered an essential candidate gene that is co-located with peak SNPs linked to these traits in M. esculenta. A review of the cassava reference genome v7.1 revealed that the SNP on chromosome 6 is in proximity to Manes.06G101600.1, a gene that regulates endodermal differentiation and root development in plants. The findings of this study provide insights into the genetic basis of plant architecture and yield in cassava. Cassava breeders could leverage this knowledge to optimize plant architecture and yield in cassava through marker-assisted selection and targeted manipulation of the candidate gene.

Keywords: manihot esculenta crantz, plant architecture, dartseq, snp markers, genome-wide association study

Procedia PDF Downloads 49

Authors: R. Sulakshana, R. Lakshmi

Abstract:

Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR associate (CRISPR/Cas) is an adaptive immunity system found in bacteria and archaea. It has been modified to serve as a potent gene editing tool. Moreover, it has found widespread use in the field of genome research because of its accessibility and low cost. Several bioinformatics methods have been created to aid in the construction of specific single guide RNA (sgRNA), which is highly active and crucial to CRISPR/Cas performance. Various Cas proteins, including Cas1, Cas2, Cas9, and Cas12, have been used to create genome engineering tools because of their programmable sequence specificity. Class 1 and 2 CRISPR/Cas systems, as well as the processes of all known Cas proteins (including Cas9 and Cas12), are discussed in this review paper. In addition, the various CRISPR methodologies and their tools so far discovered are discussed. Finally, the challenges and issues in the CRISPR system along with future works, are presented.

Keywords: gene editing tool, Cas proteins, CRISPR, guideRNA, programmable sequence

Procedia PDF Downloads 65

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

Abstract:

A huge revolution has emerged in genome engineering by the discovery of CRISPR (clustered regularly interspaced palindromic repeats) and CRISPR-associated system genes (Cas) in bacteria. The function of type II Streptococcus pyogenes (Sp) CRISPR/Cas9 system has been confirmed in various species. Other S. thermophilus (St) CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been also reported for preventing phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. The S. thermophilus CRISPR3-Cas system also acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed in the CRISPR1-Cas system. It is worth mentioning, for the effective DNA cleavage activity, RNA-guided Cas9 orthologs require their own specific PAM (protospacer adjacent motif) sequences. Activity levels are based on the sequence of the protospacer and specific combinations of favorable PAM bases. Therefore, based on the specific length and sequence of PAM followed by a constant length of target site for the three orthogonals of Cas9 protein, a well-organized procedure will be required for high-throughput and accurate mining of possible target sites in a large genomic dataset. Consequently, we created a reliable procedure to explore potential gRNA sequences for type I (Streptococcus thermophiles), II (Streptococcus pyogenes), and III (Streptococcus thermophiles) CRISPR/Cas systems. To mine CRISPR target sites, four different searching modes of sgRNA binding to target DNA strand were applied. These searching modes are as follows: i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching, and iv) paired-gRNA searching. The output of such procedure highlights the power of comparative genome mining for different CRISPR/Cas systems. This could yield a repertoire of Cas9 variants with expanded capabilities of gRNA design, and will pave the way for further advance genome and epigenome engineering.

Keywords: CRISPR/Cas systems, gRNA mining, Streptococcus pyogenes, Streptococcus thermophiles

Procedia PDF Downloads 219

Authors: Daria Grechishnikova, Maria Poptsova

Abstract:

Transposable elements play an important role in the evolution of various species from bacteria to human. Long Interspersed Nuclear Elements (LINEs) and Short Interspersed Nuclear Elements (SINEs) are two major classes of retrotransposons that occupy a considerable part of any genome and their copy numbers can range form several hundreds to a million. Both LINEs and SINEs multiply through a copy-and-paste mechanism. LINEs encode proteins, which make them capable of self-propagation while SINEs are parasitic and require the machinery of LINEs to multiply. The mechanisms how LINE and SINE RNA is recognized by the LINE-encoded reverse transcriptase (RT) remain unclear. For some SINE-LINE pairs, it was shown that they share a common 3’-end with a stem-loop structure. Majority of the SINE-LINE pairs do not have a common 3’-end. Recently we have shown that in the human genome Alu-L1 pairs have structurally similar stem-loop structure at the 3’-end. Here we extended our analysis to a wide range of species and analyzed LINEs from 161 different species from Repbase and 217 SINE sequences from SINEBase. It appeared that all of the analyzed sequences contained stem-loop structures at the 3’-end. Here we conclude that it is very likely that a common evolutionary mechanism of transposon RNA recognition requires the presence of stem-loop structures at their 3’-end.

Keywords: LINE, SINE, mechanisms of retrotransposition, retrotransposons, stem-loop, stem-loop structures, transposons

Procedia PDF Downloads 321

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

Abstract:

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

Procedia PDF Downloads 358

Authors: S. Fahiminiya, J. Nadaf, F. Rauch, L. Jerome-Majewska, J. Majewski

Abstract:

Background: Sequencing of protein coding regions of human genome (Whole Exome Sequencing; WES), has demonstrated a great success in the identification of causal mutations for several rare genetic disorders in human. Generally, most of WES studies have focused on rare variants in coding exons and splicing-sites where missense substitutions lead to the alternation of protein product. Although focusing on this category of variants has revealed the mystery behind many inherited genetic diseases in recent years, a subset of them remained still inconclusive. Here, we present the result of our WES studies where analyzing only rare variants in coding regions was not conclusive but further investigation revealed the involvement of non-coding variants and copy number variations (CNV) in etiology of the diseases. Methods: Whole exome sequencing was performed using our standard protocols at Genome Quebec Innovation Center, Montreal, Canada. All bioinformatics analyses were done using in-house WES pipeline. Results: To date, we successfully identified several disease causing mutations within gene coding regions (e.g. SCARF2: Van den Ende-Gupta syndrome and SNAP29: 22q11.2 deletion syndrome) by using WES. In addition, we showed that variants in non-coding regions and CNV have also important value and should not be ignored and/or filtered out along the way of bioinformatics analysis on WES data. For instance, in patients with osteogenesis imperfecta type V and in patients with glucocorticoid deficiency, we identified variants in 5'UTR, resulting in the production of longer or truncating non-functional proteins. Furthermore, CNVs were identified as the main cause of the diseases in patients with metaphyseal dysplasia with maxillary hypoplasia and brachydactyly and in patients with osteogenesis imperfecta type VII. Conclusions: Our study highlights the importance of considering non-coding variants and CNVs during interpretation of WES data, as they can be the only cause of disease under investigation.

Keywords: whole exome sequencing data, non-coding variants, copy number variations, rare diseases

Procedia PDF Downloads 387

Authors: Ruolan Zhou

Abstract:

Trisomy of human chromosome 21 is the primary cause of Down Syndrome (DS), and this genetic disease has significantly burdened families and countries, causing great controversy. To address this problem, the research takes an approach in exploring the relationship between genetic abnormality and this disease's symptoms, adopting several techniques, including data analysis and enrichment analysis. It also explores open-source websites, such as NCBI, DAVID, SOURCE, STRING, as well as UCSC, to complement its result. This research has analyzed the variety of genes on human chromosome 21 with simple coding, and by using analysis, it has specified the protein-coding genes, their function, and their location. By using enrichment analysis, this paper has found the abundance of keratin production-related coding-proteins on human chromosome 21. By adopting past researches, this research has attempted to disclose the relationship between trisomy of human chromosome 21 and keratin production abnormality, which might be the reason for common diseases in patients with Down Syndrome. At last, by addressing the advantage and insufficiency of this research, the discussion has provided specific directions for future research.

Keywords: Down Syndrome, protein production, genome, enrichment analysis

Procedia PDF Downloads 94

Authors: Jose A. Aznar-Moreno, Xi Jiang, Stefan Burén, Luis M. Rubio

Abstract:

Integration of prokaryotic nitrogen fixation (nif) genes into the plastid genome for expression of functional nitrogenase components could render plants capable of assimilating atmospheric N2 making their crops less dependent of nitrogen fertilizers. The nitrogenase Fe protein component (NifH) has been used as proxy for expression and targeting of Nif proteins within plant and yeast cells. Here we use tobacco plants with the Azotobacter vinelandii nifH and nifM genes integrated into the plastid genome. NifH and its maturase NifM were constitutively produced in leaves, but not roots, during light and dark periods. Nif protein expression in transplastomic plants was stable throughout development. Chloroplast NifH was soluble, but it only showed in vitro activity when isolated from leaves collected at the end of the dark period. Exposing the plant extracts to elevated temperatures precipitated NifM and apo-NifH protein devoid of [Fe4S4] clusters, dramatically increasing the specific activity of remaining NifH protein. Our data indicate that the chloroplast endogenous [Fe-S] cluster biosynthesis was insufficient for complete NifH maturation, albeit a negative effect on NifH maturation due to excess NifM in the chloroplast cannot be excluded. NifH and NifM constitutive expression in transplastomic plants did not affect any of the following traits: seed size, germination time, germination ratio, seedling growth, emergence of the cotyledon and first leaves, chlorophyll content and plant height throughout development.

Keywords: NifH, chloroplast, nitrogen fixation, crop improvement, transplastomic plants, fertilizer, biotechnology

Procedia PDF Downloads 132

Authors: Ellie Fini, Mahour Parast, Daniel Oldham, Shahrzad Hosseinnezhad

Abstract:

This paper investigates the relationship between molecular species of four different bio-based adhesives (made from Swine Manure, Miscanthus Pellet, Corn Stover, and Wood Pellet) and their rheological behavior before and after they undergo extensive oxidative aging. To study the effect of oxidative aging on the chemical structure of bio-adhesives, Infrared Attenuated Total Reflectance Spectroscopy (Fourier transform infrared) was utilised. In addition, a Drop Shape Analyser, Rotational Viscometer, and Dynamic Shear Rheometer were used to evaluate the surface properties and rheological behaviour of each bio-adhesive. Overall, bio-adhesives were found to be significantly different in terms of their ageing characteristics. Accordingly, their surface and rheological properties were found to be ranked differently before and after ageing. The results showed that the bio-adhesive from swine manure is less susceptible to aging compared to plant-based bio-oils. This can be further attributed to the chemical structure and the high lipid contents of the bio-adhesive from swine manure, making it less affected by oxidative ageing.

Keywords: bio-adhesive, rheology, bio-mass, material genome

Procedia PDF Downloads 227

Authors: Arpit Kumar Shrivastava, Subrat Kumar, Rajani Kanta Mohapatra, Priyadarshi Soumyaranjan Sahu

Abstract:

Computational approaches to predict structure, function and other biological characteristics of proteins are becoming more common in comparison to the traditional methods in drug discovery. Cryptosporidiosis is a major zoonotic diarrheal disease particularly in children, which is caused primarily by Cryptosporidium hominis and Cryptosporidium parvum. Currently, there are no vaccines for cryptosporidiosis and recommended drugs are not effective. With the availability of complete genome sequence of C. hominis, new targets have been recognized for the development of effective and better drugs and/or vaccines. We identified a unique hypothetical epitopic protein in C. hominis genome through BLASTP analysis. A 3D model of the hypothetical protein was generated using I-Tasser server through threading methodology. The quality of the model was validated through Ramachandran plot by PROCHECK server. The functional annotation of the hypothetical protein through DALI server revealed structural similarity with human Transportin 3. Phylogenetic analysis for this hypothetical protein also showed C. hominis hypothetical protein (CUV04613) was the closely related to human transportin 3 protein. The 3D protein model is further subjected to virtual screening study with inhibitors from the Zinc Database by using Dock Blaster software. Docking study reported N-(3-chlorobenzyl) ethane-1,2-diamine as the best inhibitor in terms of docking score. Docking analysis elucidated that Leu 525, Ile 526, Glu 528, Glu 529 are critical residues for ligand–receptor interactions. The molecular dynamic simulation was done to access the reliability of the binding pose of inhibitor and protein complex using GROMACS software at 10ns time point. Trajectories were analyzed at each 2.5 ns time interval, among which, H-bond with LEU-525 and GLY- 530 are significantly present in MD trajectories. Furthermore, antigenic determinants of the protein were determined with the help of DNA Star software. Our study findings showed a great potential in order to provide insights in the development of new drug(s) or vaccine(s) for control as well as prevention of cryptosporidiosis among humans and animals.

Keywords: cryptosporidium hominis, hypothetical protein, molecular docking, molecular dynamics simulation

Procedia PDF Downloads 338

Authors: Supriya Ambawat, Subaran Singh, C. Tara Satyavathi, B. S. Rajpurohit, Ummed Singh, Balraj Singh

Abstract:

Pearl millet [Pennisetum glaucum (L.) R. Br.] is an important staple food of the arid and semiarid tropical regions of Asia, Africa, and Latin America. It is rightly termed as nutricereal as it has high nutrition value and a good source of carbohydrate, protein, fat, ash, dietary fiber, potassium, magnesium, iron, zinc, etc. Pearl millet has low prolamine fraction and is gluten free which is useful for people having a gluten allergy. It has several health benefits like reduction in blood pressure, thyroid, diabe¬tes, cardiovascular and celiac diseases but its direct consumption as food has significantly declined due to several reasons. Keeping this in view, it is important to reorient the ef¬forts to generate demand through value-addition and quality improvement and create awareness on the nutritional merits of pearl millet. In India, through Indian Council of Agricultural Research-All India Coordinated Research Project on Pearl millet, multilocational coordinated trials for developed hybrids were conducted at various centers. The gene banks of pearl millet contain varieties with high levels of iron and zinc which were used to produce new pearl millet varieties with elevated iron levels bred with the high‐yielding varieties. Thus, using breeding approaches and biochemical analysis, a total of 167 hybrids and 61 varieties were identified and released for cultivation in different agro-ecological zones of the country which also includes some biofortified hybrids rich in Fe and Zn. Further, using several biotechnological interventions such as molecular markers, next-generation sequencing (NGS), association mapping, nested association mapping (NAM), MAGIC populations, genome editing, genotyping by sequencing (GBS), genome wide association studies (GWAS) advancement in millet improvement has become possible by identifying and tagging of genes underlying a trait in the genome. Using DArT markers very high density linkage maps were constructed for pearl millet. Improved HHB67 has been released using marker assisted selection (MAS) strategies, and genomic tools were used to identify Fe-Zn Quantitative Trait Loci (QTL). The draft genome sequence of millet has also opened various ways to explore pearl millet. Further, genomic positions of significantly associated simple sequence repeat (SSR) markers with iron and zinc content in the consensus map is being identified and research is in progress towards mapping QTLs for flour rancidity. The sequence information is being used to explore genes and enzymatic pathways responsible for rancidity of flour. Thus, development and application of several biotechnological approaches along with biofortification can accelerate the genetic gain targets for pearl millet improvement and help improve its quality.

Keywords: Biotechnological approaches, genomic tools, malnutrition, MAS, nutricereal, pearl millet, sequencing.

Procedia PDF Downloads 135

Authors: András Farkas, István Molnár, Jan Vrána, Veronika Burešová, Petr Cápal, András Cseh, Márta Molnár-Láng, Jaroslav Doležel

Abstract:

Species of Aegilops played a central role in the evolution of wheat and are sources of traits related to yield quality and tolerance against biotic and abiotic stresses. These wild genes and alleles are desirable to use in crop improvement programs via introgressive hybridization. However, the success of chromosome mediated gene transfer to wheat are hampered by the pour knowledge on the genome structure of Aegilops relative to wheat and by the low number of cost-effective molecular markers specific for Aegilops chromosomes. The COS markers specific for genes conserved throughout evolution in both sequence and copy number between Triticeae/Aegilops taxa and define orthologous regions, thus enabling the comparison of regions on the chromosomes of related species. The present study compared individual chromosomes of Aegilops umbellulata (UU), Ae. comosa (MM), Ae. speltoides (SS) and Ae. caudata (CC) purified by flourescent labelling with oligonucleotid SSR repeats and biparametric flow cytometry with wheat by identifying orthologous chromosomal regions by COS markers. The linear order of bin-mapped COS markers along the wheat D chromosomes was identified by the use of chromosome-specific sequence data and virtual gene order. Syntenic regions of wheat identifying genome rearrangements differentiating the U, M, S or C genomes from the D genome of wheat were detected. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species and wheat will facilitate the targeted development of new markers specific for U, M, S and C genomic regions and will contribute to the understanding of molecular processes related to the evolution of Aegilops.

Keywords: Aegilops, cos-markers, flow-sorting, wheat

Procedia PDF Downloads 467

Authors: Po-Jung Huang, Chi-Ching Lee, Bertrand Chin-Ming Tan, Yuan-Ming Yeh, Julie Lichieh Chu, Tin-Wen Chen, Cheng-Yang Lee, Ruei-Chi Gan, Hsuan Liu, Petrus Tang

Abstract:

Whole-exome sequencing focuses on the protein coding regions of disease/cancer associated genes based on a priori knowledge is the most cost-effective method to study the association between genetic alterations and disease. Recent advances in high throughput sequencing technologies and proteomic techniques has provided an opportunity to integrate genomics and proteomics, allowing readily detectable mutated peptides corresponding to mutated genes. Since sequence database search is the most widely used method for protein identification using Mass spectrometry (MS)-based proteomics technology, a mutant proteome database is required to better approximate the real protein pool to improve disease-associated mutated protein identification. Large-scale whole exome/genome sequencing studies were launched by National Cancer Institute (NCI), Broad Institute, and The Cancer Genome Atlas (TCGA), which provide not only a comprehensive report on the analysis of coding variants in diverse samples cell lines but a invaluable resource for extensive research community. No existing database is available for the collection of mutant protein sequences related to the identified variants in these studies. CMPD is designed to address this issue, serving as a bridge between genomic data and proteomic studies and focusing on protein sequence-altering variations originated from both germline and cancer-associated somatic variations.

Keywords: TCGA, cancer, mutant, proteome

Procedia PDF Downloads 559

Authors: Nemat Sokhandan Bashir

Abstract:

Threats from viruses to agricultural crops could be even larger than the losses caused by the other pathogens because, in many cases, the viral infection is latent but crucial from an epidemic point of view. Wild vegetation can be a source of many viruses that eventually find their destiny in crop plants. Although often asymptomatic in wild plants due to adaptation, they can potentially cause serious losses in crops. Therefore, exploring viruses in wild vegetation is very important. Recently, omics have been quite useful for exploring plant viruses from various plant sources, especially wild vegetation. For instance, we have discovered viruses such as Ambrossia asymptomatic virus I (AAV-1) through the application of metagenomics from Oklahoma Prairie Reserve. Accordingly, extracts from randomly-sampled plants are subjected to high speed and ultracentrifugation to separated virus-like particles (VLP), then nucleic acids in the form of DNA or RNA are extracted from such VLPs by treatment with phenol—chloroform and subsequent precipitation by ethanol. The nucleic acid preparations are separately treated with RNAse or DNAse in order to determine the genome component of VLPs. In the case of RNAs, the complementary cDNAs are synthesized before submitting to DNA sequencing. However, for VLPs with DNA contents, the procedure would be relatively straightforward without making cDNA. Because the length of the nucleic acid content of VPLs can be different, various strategies are employed to achieve sequencing. Techniques similar to so-called "chromosome walking" may be used to achieve sequences of long segments. When the nucleotide sequence data were obtained, they were subjected to BLAST analysis to determine the most related previously reported virus sequences. In one case, we determined that the novel virus was AAV-l because the sequence comparison and analysis revealed that the reads were the closest to the Indian citrus ringspot virus (ICRSV). AAV—l had an RNA genome with 7408 nucleotides in length and contained six open reading frames (ORFs). Based on phylogenies inferred from the replicase and coat protein ORFs of the virus, it was placed in the genus Mandarivirus.

Keywords: wild, plant, novel, metagenomics

Procedia PDF Downloads 42

Authors: Yi-Hao Wong, Li-Li Chan, Chee-Onn Leong, Stephen Ambu, Joon-Wah Mak, Priyasashi Sahu

Abstract:

Background: Acanthamoeba is a genus of amoebae which lives as a free-living in nature or as a human pathogen that causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic process which adds methyl groups to DNA, is used by eukaryotic cells, including several human parasites to control their gene expression. We used qPCR, siRNA gene silencing, and RNA sequencing (RNA-Seq) to study DNA-methyltransferase gene family (DNMT) in order to indicate the possibility of its involvement in programming Acanthamoeba virulence potential. Methods: A virulence-attenuated Acanthamoeba isolate (designation: ATCC; original isolate: ATCC 50492) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of DNMT between ATCC and AC/5 isolates was performed by qPCR. Silencing on DNMT gene expression in AC/5 isolate was achieved by siRNA duplex. Total RNAs extracted from ATCC, AC/5, and siRNA-treated (designation: si-146) were subjected to RNA-Seq for comparative transcriptomic analysis in order to identify the genome-wide effect of DNMT in regulating Acanthamoeba gene expression. qPCR was performed to validate the RNA-Seq results. Results: Physiological and cytophatic assays demonstrated an increased in virulence potential of AC/5 isolate after mouse passages. DNMT gene expression was significantly higher in AC/5 compared to ATCC isolate (p ≤ 0.01) by qPCR. si-146 duplex reduced DNMT gene expression in AC/5 isolate by 30%. Comparative transcriptome analysis identified the differentially expressed genes, with 3768 genes in AC/5 vs ATCC isolate; 2102 genes in si-146 vs AC/5 isolate and 3422 genes in si-146 vs ATCC isolate, respectively (fold-change of ≥ 2 or ≤ 0.5, p-value adjusted (padj) < 0.05). Of these, 840 and 1262 genes were upregulated and downregulated, respectively, in si-146 vs AC/5 isolate. Eukaryotic orthologous group (KOG) assignments revealed a higher percentage of downregulated gene expression in si-146 compared to AC/5 isolate, were related to posttranslational modification, signal transduction and energy production. Gene Ontology (GO) terms for those downregulated genes shown were associated with transport activity, oxidation-reduction process, and metabolic process. Among these downregulated genes were putative genes encoded for heat shock proteins, transporters, ubiquitin-related proteins, proteins for vesicular trafficking (small GTPases), and oxidoreductases. Functional analysis of similar predicted proteins had been described in other parasitic protozoa for their survival and pathogenicity. Decreased expression of these genes in si146-treated isolate may account in part for Acanthamoeba reduced pathogenicity. qPCR on 6 selected genes upregulated in AC/5 compared to ATCC isolate corroborated the RNA sequencing findings, indicating a good concordance between these two analyses. Conclusion: To the best of our knowledge, this study represents the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba spp. The present data indicate that DNA methylation has substantial effect on global gene expression, allowing further dissection of the genome-wide effects of DNA-methyltransferase gene in regulating Acanthamoeba pathogenicity.

Keywords: Acanthamoeba, DNA methylation, RNA sequencing, virulence

Procedia PDF Downloads 168

Authors: Ewelina Wisnik, Zsolt Regdon, Kinga Chmielewska, Laszlo Virag, Agnieszka Robaszkiewicz

Abstract:

Apart from protecting genome, PARP1 has been documented to regulate many intracellular processes inter alia gene transcription by physically interacting with chromatin bound proteins and by their ADP-ribosylation. Our recent findings indicate that expression of PARP1 decreases during the differentiation of human CD34+ hematopoietic stem cells to monocytes as a consequence of differentiation-associated cell growth arrest and formation of E2F4-RBL2-HDAC1-SWI/SNF repressive complex at the promoter of this gene. Since the RBL2 complexes repress genes in a E2F-dependent manner and are widespread in the genome in G0 arrested cells, we asked (a) if RBL2 directly contributes to defining monocyte phenotype and function by targeting gene promoters and (b) if RBL2 controls gene transcription indirectly by repressing PARP1. For identification of genes controlled by RBL2 and/or PARP1,we used primer libraries for surface receptors and TLR signaling mediators, genes were silenced by siRNA or shRNA, analysis of gene promoter occupation by selected proteins was carried out by ChIP-qPCR, while statistical analysis in GraphPad Prism 5 and STATISTICA, ChIP-Seq data were analysed in Galaxy 2.5.0.0. On the list of 28 genes regulated by RBL2, we identified only four solely repressed by RBL2-E2F4-HDAC1-BRM complex. Surprisingly, 24 out of 28 emerged genes controlled by RBL2 were co-regulated by PARP1 in six different manners. In one mode of RBL2/PARP1 co-operation, represented by MAP2K6 and MAPK3, PARP1 was found to associate with gene promoters upon RBL2 silencing, which was previously shown to restore PARP1 expression in monocytes. PARP1 effect on gene transcription was observed only in the presence of active EP300, which acetylated gene promoters and activated transcription. Further analysis revealed that PARP1 binding to MA2K6 and MAPK3 promoters enabled recruitment of EP300 in monocytes, while in proliferating cancer cell lines, which actively transcribe PARP1, this protein maintained EP300 at the promoters of MA2K6 and MAPK3. Genome-wide analysis revealed a similar distribution of PARP1 and EP300 around transcription start sites and the co-occupancy of some gene promoters by PARP1 and EP300 in cancer cells. Here, we described a new RBL2/PARP1/EP300 axis which controls gene transcription regardless of the cell type. In this model cell, cycle-dependent transcription of PARP1 regulates expression of some genes repressed by RBL2 upon cell cycle limitation. Thus, RBL2 may indirectly regulate transcription of some genes by controlling the expression of EP300-recruiting PARP1. Acknowledgement: This work was financed by Polish National Science Centre grants nr DEC-2013/11/D/NZ2/00033 and DEC-2015/19/N/NZ2/01735. L.V. is funded by the National Research, Development and Innovation Office grants GINOP-2.3.2-15-2016-00020 TUMORDNS, GINOP-2.3.2-15-2016-00048-STAYALIVE and OTKA K112336. AR is supported by Polish Ministry of Science and Higher Education 776/STYP/11/2016.

Keywords: retinoblastoma transcriptional co-repressor like 2 (RBL2), poly(ADP-ribose) polymerase 1 (PARP1), E1A binding protein p300 (EP300), monocytes

Procedia PDF Downloads 174

Authors: Igor V. Grigoriev

Abstract:

Understanding of biological processes encoded in fungi is instrumental in addressing future food, feed, and energy demands of the growing human population. Genomics is a powerful and quickly evolving tool to understand these processes. The Fungal Genomics Program of the US Department of Energy Joint Genome Institute (JGI) partners with researchers around the world to explore fungi in several large scale genomics projects, changing the fungal genomics landscape. The key trends of these changes include: (i) rapidly increasing scale of sequencing and analysis, (ii) developing approaches to go beyond culturable fungi and explore fungal ‘dark matter,’ or unculturables, and (iii) functional genomics and multi-omics data integration. Power of comparative genomics has been recently demonstrated in several JGI projects targeting mycorrhizae, plant pathogens, wood decay fungi, and sugar fermenting yeasts. The largest JGI project ‘1000 Fungal Genomes’ aims at exploring the diversity across the Fungal Tree of Life in order to better understand fungal evolution and to build a catalogue of genes, enzymes, and pathways for biotechnological applications. At this point, at least 65% of over 700 known families have one or more reference genomes sequenced, enabling metagenomics studies of microbial communities and their interactions with plants. For many of the remaining families no representative species are available from culture collections. To sequence genomes of unculturable fungi two approaches have been developed: (a) sequencing DNA from fruiting bodies of ‘macro’ and (b) single cell genomics using fungal spores. The latter has been tested using zoospores from the early diverging fungi and resulted in several near-complete genomes from underexplored branches of the Fungal Tree, including the first genomes of Zoopagomycotina. Genome sequence serves as a reference for transcriptomics studies, the first step towards functional genomics. In the JGI fungal mini-ENCODE project transcriptomes of the model fungus Neurospora crassa grown on a spectrum of carbon sources have been collected to build regulatory gene networks. Epigenomics is another tool to understand gene regulation and recently introduced single molecule sequencing platforms not only provide better genome assemblies but can also detect DNA modifications. For example, 6mC methylome was surveyed across many diverse fungi and the highest among Eukaryota levels of 6mC methylation has been reported. Finally, data production at such scale requires data integration to enable efficient data analysis. Over 700 fungal genomes and other -omes have been integrated in JGI MycoCosm portal and equipped with comparative genomics tools to enable researchers addressing a broad spectrum of biological questions and applications for bioenergy and biotechnology.

Keywords: fungal genomics, single cell genomics, DNA methylation, comparative genomics

Procedia PDF Downloads 179

Authors: Mudawamah Mudawamah, Muhammad Z. Fadli, Gatot Ciptadi, Aulanni’am

Abstract:

Canary breeders have spread throughout Indonesian regions for the low-middle society and become an income source for them. The interesting phenomenon of the canary market is the feather colours become one of determining factor for the price. The advantages of this research were contributed to the molecular database as a base of selection and mating for the Indonesia canary breeder. The research method was experiment with the genome obtained from canary blood isolation. The genome did the PCR amplification with PMEL marker followed by sequencing. Canaries were used 24 heads of light and dark colour feathers. Research data analyses used BioEdit and Network 4.6.0.0 software. The results showed that all samples were amplification with PMEL gene with 500 bp fragment length. In base sequence of 40 was found Cytosine(C) in the light colour canaries, while the dark colour canaries was obtained Thymine (T) in same base sequence. Sequence results had 286-415 bp fragment and 10 haplotypes. The conclusions were the PMEL gene (gene of white pigment) was likely to be used PMEL gene to detect molecular genetic variation of dark and light colour feather.

Keywords: canary, haplotype, PMEL, sequence

Procedia PDF Downloads 205

Authors: Sridhar A. Malkaram, Tamer E. Fandy

Abstract:

Purpose: 5-Azacytidine (5AC) and decitabine (DC) are DNA hypomethylating agents. We recently demonstrated that both drugs increase the enzymatic activity of the histone deacetylase enzyme SIRT6. Accordingly, we are comparing the changes H3K9 acetylation changes in the whole genome induced by both drugs using leukemia cells. Description of Methods & Materials: Mononuclear cells from the bone marrow of six de-identified naive acute myeloid leukemia (AML) patients were cultured with either 500 nM of DC or 5AC for 72 h followed by ChIP-Seq analysis using a ChIP-validated acetylated-H3K9 (H3K9ac) antibody. Chip-Seq libraries were prepared from treated and untreated cells using SMARTer ThruPLEX DNA- seq kit (Takara Bio, USA) according to the manufacturer’s instructions. Libraries were purified and size-selected with AMPure XP beads at 1:1 (v/v) ratio. All libraries were pooled prior to sequencing on an Illumina HiSeq 1500. The dual-indexed single-read Rapid Run was performed with 1x120 cycles at 5 pM final concentration of the library pool. Sequence reads with average Phred quality < 20, with length < 35bp, PCR duplicates, and those aligning to blacklisted regions of the genome were filtered out using Trim Galore v0.4.4 and cutadapt v1.18. Reads were aligned to the reference human genome (hg38) using Bowtie v2.3.4.1 in end-to-end alignment mode. H3K9ac enriched (peak) regions were identified using diffReps v1.55.4 software using input samples for background correction. The statistical significance of differential peak counts was assessed using a negative binomial test using all individuals as replicates. Data & Results: The data from the six patients showed significant (Padj<0.05) acetylation changes at 925 loci after 5AC treatment versus 182 loci after DC treatment. Both drugs induced H3K9 acetylation changes at different chromosomal regions, including promoters, coding exons, introns, and distal intergenic regions. Ten common genes showed H3K9 acetylation changes by both drugs. Approximately 84% of the genes showed an H3K9 acetylation decrease by 5AC versus 54% only by DC. Figures 1 and 2 show the heatmaps for the top 100 genes and the 99 genes showing H3K9 acetylation decrease after 5AC treatment and DC treatment, respectively. Conclusion: Despite the similarity in hypomethylating activity and chemical structure, the effect of both drugs on H3K9 acetylation change was significantly different. More changes in H3K9 acetylation were observed after 5 AC treatments compared to DC. The impact of these changes on gene expression and the clinical efficacy of these drugs requires further investigation.

Keywords: DNA methylation, leukemia, decitabine, 5-Azacytidine, epigenetics

Procedia PDF Downloads 116

Authors: Ante Turudic, Filip Varga, Zlatko Liber, Jernej Jakse, Zlatko Satovic, Ivan Radosavljevic, Martina Grdisa

Abstract:

Microsatellites (SSRs) are highly informative repetitive sequences of 2-6 base pairs, which are the most used molecular markers in assessing the genetic diversity of plant species. Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip) is an outcrossing diploid (2n = 18) endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repetitiveness and large size of the genome (haploid genome size of 9,58 pg), previous attempts to develop microsatellite markers using the standard methods were unsuccessful. A next-generation sequencing (NGS) approach was applied on genomic DNA extracted from fresh leaves of Dalmatian pyrethrum. The sequencing was conducted using NovaSeq6000 Illumina sequencer, after which almost 400 million high-quality paired-end reads were obtained, with a read length of 150 base pairs. Short reads were assembled by combining two approaches; (1) de-novo assembly and (2) joining of overlapped pair-end reads. In total, 6.909.675 contigs were obtained, with the contig average length of 249 base pairs. Of the resulting contigs, 31.380 contained one or multiple microsatellite sequences, in total 35.556 microsatellite loci were identified. Out of detected microsatellites, dinucleotide repeats were the most frequent, accounting for more than half of all microsatellites identifies (21,212; 59.7%), followed by trinucleotide repeats (9,204; 25.9%). Tetra-, penta- and hexanucleotides had similar frequency of 1,822 (5.1%), 1,472 (4.1%), and 1,846 (5.2%), respectively. Contigs containing microsatellites were further filtered by SSR pattern type, transposon occurrences, assembly characteristics, GC content, and the number of occurrences against the draft genome of T. cinerariifolium published previously. After the selection process, 50 microsatellite loci were used for primer design. Designed primers were tested on samples from five distinct populations, and 25 of them showed a high degree of polymorphism. The selected loci were then genotyped on 20 samples belonging to one population resulting in 17 microsatellite markers. Availability of codominant SSR markers will significantly improve the knowledge on population genetic diversity and structure as well as complex genetics and biochemistry of this species. Acknowledgment: This work has been fully supported by the Croatian Science Foundation under the project ‘Genetic background of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir/ Sch. Bip.) insecticidal potential’ - (PyrDiv) (IP-06-2016-9034).

Keywords: genome assembly, NGS, SSR, Tanacetum cinerariifolium

Procedia PDF Downloads 99

Authors: Soultana Konstantinidou, Tiziana Schmidt, Elena Landi, Alessandro De Carli, Giovanni Maltinti, Darius Witt, Alicja Dziadosz, Agnieszka Lindstaedt, Michele Lai, Mauro Pistello, Valentina Cappello, Luciana Dente, Chiara Gabellini, Piotr Barski, Vittoria Raffa

Abstract:

CRISPR/Cas9 technology has gained the interest of researchers in the field of biotechnology for genome editing. Since its discovery as a microbial adaptive immune defense, this system has been widely adopted and is acknowledged for having a variety of applications. However, critical barriers related to safety and delivery are persisting. Here, we propose a new concept of genome engineering, which is based on a nano-formulation of Cas9. The Cas9 enzyme was conjugated to a gold nanoparticle (AuNP-Cas9). The AuNP-Cas9 maintained its cleavage efficiency in vitro, to the same extent as the ribonucleoprotein, including non-conjugated Cas9 enzyme, and showed high gene editing efficiency in vivo in zebrafish embryos. Since CRISPR/Cas9 technology is extensively used in cancer research, melanoma was selected as a validation target. Cell studies were performed in A375 human melanoma cells. Particles per se had no impact on cell metabolism and proliferation. Intriguingly, the AuNP-Cas9 internalized spontaneously in cells and localized as a single particle in the cytoplasm and organelles. More importantly, the AuNP-Cas9 showed a high nuclear localization signal. The AuNP-Cas9, overcoming the delivery difficulties of Cas9, could be used in cellular biology and localization studies. Taking advantage of the plasmonic properties of gold nanoparticles, this technology could potentially be a bio-tool for combining gene editing and photothermal therapy in cancer cells. Further work will be focused on intracellular interactions of the nano-formulation and characterization of the optical properties.

Keywords: CRISPR/Cas9, gene editing, gold nanoparticles, nanotechnology

Procedia PDF Downloads 71

Authors: Feng-Sheng Wang, Chao-Ting Cheng

Abstract:

Developing a drug from conception to launch is costly and time-consuming. Computer-aided methods can reduce research costs and accelerate the development process during the early drug discovery and development stages. This study developed a fuzzy multi-objective hierarchical optimization framework for identifying potential anticancer targets in a metabolic model. First, RNA-seq expression data of colorectal cancer samples and their healthy counterparts were used to reconstruct tissue-specific genome-scale metabolic models. The aim of the optimization framework was to identify anticancer targets that lead to cancer cell death and evaluate metabolic flux perturbations in normal cells that have been caused by cancer treatment. Four objectives were established in the optimization framework to evaluate the mortality of cancer cells for treatment and to minimize side effects causing toxicity-induced tumorigenesis on normal cells and smaller metabolic perturbations. Through fuzzy set theory, a multiobjective optimization problem was converted into a trilevel maximizing decision-making (MDM) problem. The applied nested hybrid differential evolution was applied to solve the trilevel MDM problem using two nutrient media to identify anticancer targets in the genome-scale metabolic model of colorectal cancer, respectively. Using Dulbecco’s Modified Eagle Medium (DMEM), the computational results reveal that the identified anticancer targets were mostly involved in cholesterol biosynthesis, pyrimidine and purine metabolisms, glycerophospholipid biosynthetic pathway and sphingolipid pathway. However, using Ham’s medium, the genes involved in cholesterol biosynthesis were unidentifiable. A comparison of the uptake reactions for the DMEM and Ham’s medium revealed that no cholesterol uptake reaction was included in DMEM. Two additional media, i.e., a cholesterol uptake reaction was included in DMEM and excluded in HAM, were respectively used to investigate the relationship of tumor cell growth with nutrient components and anticancer target genes. The genes involved in the cholesterol biosynthesis were also revealed to be determinable if a cholesterol uptake reaction was not induced when the cells were in the culture medium. However, the genes involved in cholesterol biosynthesis became unidentifiable if such a reaction was induced.

Keywords: Cancer metabolism, genome-scale metabolic model, constraint-based model, multilevel optimization, fuzzy optimization, hybrid differential evolution

Procedia PDF Downloads 50

Authors: Ahmed M. Debela, Mayreli Ortiz , Ciara K. O´Sullivan

Abstract:

The completion of the human genome Project (HGP) has paved the way for mapping the diversity in the overall genome sequence which helps to understand the genetic causes of inherited diseases and susceptibility to drugs or environmental toxins. Arrayed primer extension (APEX) is a microarray based minisequencing strategy for screening disease causing mutations. It is derived from Sanger DNA sequencing and uses fluorescently dideoxynucleotides (ddNTPs) for termination of a growing DNA strand from a primer with its 3´- end designed immediately upstream of a site where single nucleotide polymorphism (SNP) occurs. The use of DNA polymerase offers a very high accuracy and specificity to APEX which in turn happens to be a method of choice for multiplex SNP detection. Coupling the high specificity of this method with the high sensitivity, low cost and compatibility for miniaturization of electrochemical techniques would offer an excellent platform for detection of mutation as well as sequencing of DNA templates. We are developing an electrochemical APEX for the analysis of SNPs found in the MYH7 gene for group of cardiomyopathy patients. ddNTPs were labeled with four different redox active compounds with four distinct potentials. Thiolated oligonucleotide probes were immobilised on gold and glassy carbon substrates which are followed by hybridisation with complementary target DNA just adjacent to the base to be extended by polymerase. Electrochemical interrogation was performed after the incorporation of the redox labelled dedioxynucleotide. The work involved the synthesis and characterisation of the redox labelled ddNTPs, optimisation and characterisation of surface functionalisation strategies and the nucleotide incorporation assays.

Keywords: array based primer extension, labelled ddNTPs, electrochemical, mutations

Procedia PDF Downloads 219

Authors: Pavlina Capkova, Josef Srovnal, Vera Becvarova, Marie Trkova, Zuzana Capkova, Andrea Stefekova, Vaclava Curtisova, Alena Santava, Sarka Vejvalkova, Katerina Adamova, Radek Vodicka

Abstract:

ASDs are heterogeneous and complex developmental diseases with a significant genetic background. Recurrent CNVs are known to be a frequent cause of ASD. These CNVs can have, however, a variable expressivity which results in a spectrum of phenotypes from asymptomatic to ID/DD/ASD. ASD is associated with ID in ~75% individuals. Various platforms are used to detect pathogenic mutations in the genome of these patients. The performed study is focused on a determination of the frequency of pathogenic mutations in a group of ASD patients and a group of ID/DD patients using various strategies along with a comparison of their detection rate. The possible role of the origin of these mutations in aetiology of ASD was assessed. The study included 35 individuals with ASD and 68 individuals with ID/DD (64 males and 39 females in total), who underwent rigorous genetic, neurological and psychological examinations. Screening for pathogenic mutations involved karyotyping, screening for FMR1 mutations and for metabolic disorders, a targeted MLPA test with probe mixes Telomeres 3 and 5, Microdeletion 1 and 2, Autism 1, MRX and a chromosomal microarray analysis (CMA) (Illumina or Affymetrix). Chromosomal aberrations were revealed in 7 (1 in the ASD group) individuals by karyotyping. FMR1 mutations were discovered in 3 (1 in the ASD group) individuals. The detection rate of pathogenic mutations in ASD patients with a normal karyotype was 15.15% by MLPA and CMA. The frequencies of the pathogenic mutations were 25.0% by MLPA and 35.0% by CMA in ID/DD patients with a normal karyotype. CNVs inherited from asymptomatic parents were more abundant than de novo changes in ASD patients (11.43% vs. 5.71%) in contrast to the ID/DD group where de novo mutations prevailed over inherited ones (26.47% vs. 16.18%). ASD patients shared more frequently their mutations with their fathers than patients from ID/DD group (8.57% vs. 1.47%). Maternally inherited mutations predominated in the ID/DD group in comparison with the ASD group (14.7% vs. 2.86 %). CNVs of an unknown significance were found in 10 patients by CMA and in 3 patients by MLPA. Although the detection rate is the highest when using CMA, recurrent CNVs can be easily detected by MLPA. CMA proved to be more efficient in the ID/DD group where a larger spectrum of rare pathogenic CNVs was revealed. This study determined that maternally inherited highly penetrant mutations and de novo mutations more often resulted in ID/DD without ASD in patients. The paternally inherited mutations could be, however, a source of the greater variability in the genome of the ASD patients and contribute to the polygenic character of the inheritance of ASD. As the number of the subjects in the group is limited, a larger cohort is needed to confirm this conclusion. Inherited CNVs have a role in aetiology of ASD possibly in combination with additional genetic factors - the mutations elsewhere in the genome. The identification of these interactions constitutes a challenge for the future. Supported by MH CZ – DRO (FNOl, 00098892), IGA UP LF_2016_010, TACR TE02000058 and NPU LO1304.

Keywords: autistic spectrum disorders, copy number variant, chromosomal microarray, intellectual disability, karyotyping, MLPA, multiplex ligation-dependent probe amplification

Procedia PDF Downloads 325

Authors: Yunfeng Shan, Xiaoliang Wang, Youjun Zhou

Abstract:

Whole-genome data from two lungfish species, along with other species, present a valuable opportunity to re-investigate the longstanding debate regarding the evolutionary relationships among tetrapods, lungfishes, and coelacanths. However, the use of bootstrap support has become outdated for large-scale phylogenomic data. Without robust phylogenetic support, the phylogenetic trees become meaningless. Therefore, it is necessary to re-evaluate the phylogenies of tetrapods, lungfishes, and coelacanths using novel measures of phylogenetic support specifically designed for phylogenomic data, as the previous phylogenies were based on 100% bootstrap support. Our findings consistently provide strong evidence favoring lungfish as the closest living relative of tetrapods. This conclusion is based on high internode certainty, relative gene support, and high gene concordance factor. The evidence stems from five previous datasets derived from lungfish transcriptomes. These results yield fresh insights into the three hypotheses regarding the phylogenies of tetrapods, lungfishes, and coelacanths. Importantly, these hypotheses are not mere conjectures but are substantiated by a significant number of genes. Analyzing real biological data further demonstrates that the inclusion of additional taxa leads to more diverse tree topologies. Consequently, gene trees and species trees may not be identical even when whole-genome sequencing data is utilized. However, it is worth noting that many gene trees can accurately reflect the species tree if an appropriate number of taxa, typically ranging from six to ten, are sampled. Therefore, it is crucial to carefully select the number of taxa and an appropriate outgroup, such as slow-evolving species, while excluding fast-evolving taxa as outgroups to mitigate the adverse effects of long-branch attraction and achieve an accurate reconstruction of the species tree. This is particularly important as more whole-genome sequencing data becomes available.

Keywords: novel measures of phylogenetic support for phylogenomic data, gene concordance factor confidence, relative gene support, internode certainty, origin of tetrapods

Procedia PDF Downloads 27

Authors: Chamilani Nikapitiya, Mahanama De Zoysa, Jehee Lee

Abstract:

Most of the bacterial diseases are associated with high mortalities in aquaculture species and causing huge economic losses. Different approaches have been taken to prevent or control of bacterial diseases including use of vaccines, probiotics, chemotherapy, water quality management, etc. Antibiotics are widely applying as chemotherapy to control bacterial diseases, however, it has been shown that frequent use of antibiotics is favored to develop multi-drug resistance bacteria. Therefore, phages and phage encoded lytic proteins are known to be one of the most promising alternatives for antibiotics to avoid the emergence of antibiotic-resistant bacteria. We isolated and characterized the two lytic phages, namely pAh-1 and pAs-1 against pathogenic Aeromonas hydrophila and Aeromonas salmonicida, respectively. Morphological characteristics were analyzed by Transmission electron microscopy (TEM) and host strain specificities were tested with Aeromonas and other closely related bacterial strains. TEM analysis revealed that both pAh-1 and pAsm-1 are composed of an icosahedral head and a segmented tail, and we suggest that, they are new members of Myoviridae family. Genome sizes of isolated phages were estimated by restriction enzyme digestion of genomic DNA using selected endonucleases followed by agarose gel electrophoresis. Estimated genome size of pAh-1 and pAs-1 were approximately 64 Kbp and 120 Kbp, respectively. Both pAh-1 and pAs-1 have shown narrow host specificity. Moreover, protective effects of phage therapy against fish pathogenic A. hydrophila were investigated in zebrafish model. The survival rate was 40% higher when zebrafish received intra-peritoneal injection (i.p.) of pAh-1 were simultaneously challenge A. hydrophila (2 x 106 CFU/fish) compared to that without phage treatment. Overall results suggest that both pAh-1 and pAs-1 can be used as a potential phage therapy to control Aeromonas infections in aquaculture.

Keywords: Aeromonas infections, antibiotic resistance, bacteriophage, bio-control, lytic phage

Procedia PDF Downloads 170

Authors: Vyacheslav Furtak, Merja Roivainen, Olga Mirochnichenko, Majid Laassri, Bella Bidzhieva, Tatiana Zagorodnyaya, Vladimir Chizhikov, Konstantin Chumakov

Abstract:

Molecular epidemiology and environmental surveillance are parts of a rational strategy to control infectious diseases. They have been widely used in the worldwide campaign to eradicate poliomyelitis, which otherwise would be complicated by the inability to rapidly respond to outbreaks and determine sources of the infection. The conventional scheme involves isolation of viruses from patients and the environment, followed by their identification by nucleotide sequences analysis to determine phylogenetic relationships. This is a tedious and time-consuming process that yields definitive results when it may be too late to implement countermeasures. Because of the difficulty of high-throughput full-genome sequencing, most such studies are conducted by sequencing only capsid genes or their parts. Therefore the important information about the contribution of other parts of the genome and inter- and intra-species recombination to viral evolution is not captured. Here we propose a new approach based on the rapid concentration of sewage samples with tangential flow filtration followed by deep sequencing and reconstruction of nucleotide sequences of viruses present in the samples. The entire nucleic acids content of each sample is sequenced, thus preserving in digital format the complete spectrum of viruses. A set of rapid algorithms was developed to separate deep sequence reads into discrete populations corresponding to each virus and assemble them into full-length consensus contigs, as well as to generate a complete profile of sequence heterogeneities in each of them. This provides an effective approach to study molecular epidemiology and evolution of natural viral populations.

Keywords: poliovirus, eradication, environmental surveillance, laboratory diagnosis

Procedia PDF Downloads 249

Authors: Hamza Zidoum

Abstract:

This paper investigates the impact of human genetic variation on the function of human proteins using machine-learning algorithms. Single-Nucleotide Polymorphism represents the most common form of human genome variation. We focus on the single amino-acid polymorphism located in the coding region as they can affect the protein function leading to pathologic phenotypic change. We use several supervised Machine Learning methods to identify structural properties correlated with increased risk of the missense mutation being damaging. SVM associated with Principal Component Analysis give the best performance.

Keywords: single-nucleotide polymorphism, machine learning, feature selection, SVM

Procedia PDF Downloads 346

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

Abstract:

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

Procedia PDF Downloads 284

Authors: Jingyuan Hu, Zhandong Liu

Abstract:

CRISPR genome editing technology has transformed molecular biology by accurately targeting and altering an organism’s DNA. Despite the state-of-art precision of CRISPR genome editing, the imprecise mutation outcome and off-target effects present considerable risk, potentially leading to unintended genetic changes. Targeted deep sequencing, combined with bioinformatics sequence alignment, can detect such unwanted mutations. Nevertheless, the classical method, Needleman-Wunsch (NW) algorithm may produce false alignment outcomes, resulting in inaccurate mutation identification. The key to precisely identifying CRISPR-induced mutations lies in determining optimal parameters for the sequence alignment algorithm. Hidden Markov models (HMM) are ideally suited for this task, offering flexibility across CRISPR systems by leveraging forward-backward algorithms for parameter estimation. In this study, we introduce CRISPR-HMM, a statistical software to precisely call CRISPR-induced mutations. We demonstrate that the software significantly improves precision in identifying CRISPR-induced mutations compared to NW-based alignment, thereby enhancing the overall understanding of the CRISPR gene-editing process.

Keywords: CRISPR, HMM, sequence alignment, gene editing

Procedia PDF Downloads 16