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Asset Tag: Whole Genome Sequencing,

July 7, 2019

The draft genome sequence of Pectobacterium carotovorum subsp. actinidiae KKH3 that infects kiwi plant and potential bioconversion applications

Pectobacterium carotovorum subsp. actinidiae KKH3 is an Enterobacteriaceae bacterial pathogen that infects kiwi plants, causing canker-like symptoms that pose a threat to the kiwifruit industry. Because the strain was originally isolated from woody plants and possesses numerous plant cell wall-degrading enzymes, this draft genome report provides insight into possible bioconversion applications, as well as a better understanding of this important plant pathogen.

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July 7, 2019

Complete genome sequence of the Vibrio vulnificus strain VV2014DJH, a human-pathogenic bacterium isolated from a death case in China.

Vibrio vulnificus, an opportunistic pathogen, is the causative agent of life-threatening septicemia and severe wound infections. However, the pathogenicity and virulence factors of V. vulnificus are not fully understood. Here we report the complete genome sequence of V. vulnificus VV2014DJH, which was isolated from a death case.The genome of the V. vulnificus VV2014DJH contains two circular chromosomes with a mean G+C content of 46.8%, but does not consists of any plasmids. The chromosome I and chromosome II consist of 3,303,590 and 1,770,972 bp, respectively. In addition, the genome consists of 4617 protein coding genes, 172 RNA genes and type I, II and III secretion systems were predicted.In this study, the genomic information of the V. vulnificus VV2014DJH has been described. The information would contribute to the increasing scope and depth of Vibrio genome database, and provide insights into the pathogenicity and virulence factors of V. vulnificus.

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July 7, 2019

Unlocking the biological potential of Euglena gracilis: evolution, cell biology and significance to parasitism

Photosynthetic euglenids are major components of aquatic ecosystems and relatives of trypanosomes. Euglena gracilis has considerable biotechnological potential and great adaptability, but exploitation remains hampered by the absence of a comprehensive gene catalogue. We address this by genome, RNA and protein sequencing: the E. gracilis genome is >2Gb, with 36,526 predicted proteins. Large lineage-specific paralog families are present, with evidence for flexibility in environmental monitoring, divergent mechanisms for metabolic control, and novel solutions for adaptation to extreme environments. Contributions from photosynthetic eukaryotes to the nuclear genome, consistent with the shopping bag model are found, together with transitions between kinetoplastid and canonical systems. Control of protein expression is almost exclusively post-transcriptional. These data are a major advance in understanding the nuclear genomes of euglenids and provide a platform for investigating the contributions of E. gracilis and its relatives to the biosphere.

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July 7, 2019

The plastid genome in Cladophorales green algae is encoded by hairpin chromosomes.

Virtually all plastid (chloroplast) genomes are circular double-stranded DNA molecules, typically between 100 and 200 kb in size and encoding circa 80-250 genes. Exceptions to this universal plastid genome architecture are very few and include the dinoflagellates, where genes are located on DNA minicircles. Here we report on the highly deviant chloroplast genome of Cladophorales green algae, which is entirely fragmented into hairpin chromosomes. Short- and long-read high-throughput sequencing of DNA and RNA demonstrated that the chloroplast genes of Boodlea composita are encoded on 1- to 7-kb DNA contigs with an exceptionally high GC content, each containing a long inverted repeat with one or two protein-coding genes and conserved non-coding regions putatively involved in replication and/or expression. We propose that these contigs correspond to linear single-stranded DNA molecules that fold onto themselves to form hairpin chromosomes. The Boodlea chloroplast genes are highly divergent from their corresponding orthologs, and display an alternative genetic code. The origin of this highly deviant chloroplast genome most likely occurred before the emergence of the Cladophorales, and coincided with an elevated transfer of chloroplast genes to the nucleus. A chloroplast genome that is composed only of linear DNA molecules is unprecedented among eukaryotes, and highlights unexpected variation in plastid genome architecture. Copyright © 2017 Elsevier Ltd. All rights reserved.

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July 7, 2019

The complete mitochondrial genome of Wonwhang (Pyrus pyrifolia)

This is a de novo assembly and annotation of a complete mitochondrial genome from Pyrus pyrifolia in the family Rosaceae. The complete mitochondrial genome of P. pyrifolia was assembled from PacBio RSII P6-C4 sequencing reads. The circular genome was 458,873?bp in length, containing 39 protein-coding genes, 23 tRNA genes and three rRNA genes. The nucleotide composition was A (27.5%), T (27.3%), G (22.6%) and C (22.6%) with GC content of 45.2%. Most of protein-coding genes use the canonical start codon ATG, whereas nad1, cox1, matR and rps4 use ACG, mttB uses ATT, rpl16 and rps19 uses GTG. The stop codon is also common in all mitochondrial genes. The phylogenetic analysis showed that P. pyrifolia was clustered with the Malus of Rosaceae family. Maximum-likelihood analysis suggests a clear relationship of Rosids and Asterids, which support the traditional classification.

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July 7, 2019

Genomic clues to the parental origin of the wild flowering cherry Prunus yedoensis var. nudiflora (Rosaceae)

Prunus yedoensis Matsumura is one of the popular ornamental flowering cherry trees native to northeastern Asia, and its wild populations have only been found on Jeju Island, Korea. Previous studies suggested that wild P. yedoensis (P. yedoensis var. nudiflora) is a hybrid species; however, there is no solid evidence on its exact parental origin and genomic organization. In this study, we developed a total of 38 nuclear gene-based DNA markers that can be universally amplifiable in the Prunus species using 586 Prunus Conserved Orthologous Gene Set (Prunus COS). Using the Prunus COS markers, we investigated the genetic structure of wild P. yedoensis populations and evaluated the putative parental species of wild P. yedoensis. Population structure and phylogenetic analysis of 73 wild P. yedoensis accessions and 54 accessions of other Prunus species revealed that the wild P. yedoensis on Jeju Island is a natural homoploid hybrid. Sequence-level comparison of Prunus COS markers between species suggested that wild P. yedoensis might originate from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura. Moreover, approximately 81% of the wild P. yedoensis accessions examined were likely F1 hybrids, whereas the remaining 19% were backcross hybrids resulting from additional asymmetric introgression of parental genotypes. These findings suggest that complex hybridization of the Prunus species on Jeju Island can produce a range of variable hybrid offspring. Overall, this study makes a significant contribution to address issues of the origin, nomenclature, and genetic relationship of ornamental P. yedoensis.

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July 7, 2019

Complete genome sequence of Spirosoma pulveris JSH 5-14 T, a bacterium isolated from a dust sample

Dust particles from the deserts and semiarid lands in northern China cause pollution that increase the burden of allergic disease particularly in the urban population of East Asia. Dust particles that carried with windstorm are associated with microbial populations, which include virus, bacteria, and fungi. Spirosoma pulveris JSH 5-14T isolated from the gamma ray-irradiated dust sample collected at Nonsan, Chungnam province, South Korea and showed resistance against gamma and UV radiation. We carried out the whole genome sequencing to understand insight of radiation resistance and their mechanisms of survival. The whole genome of strain JSH 5-14T is comprised of 7,188,680 bp (G+C content of 50.50%) including 5,896 protein-coding genes and 52 RNA genes. The genome analysis of strain JSH 5-14T showed the presence of several genes involved in DNA repair pathways and defense mechanism against irradiation. In this study, we discuss the implication of such findings concerning other radiation resistant bacteria.

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July 7, 2019

Mechanisms of adaptive divergence and speciation in Littorina saxatilis: Integrating knowledge from ecology and genetics with new data emerging from genomic studies

New opportunities to understand marine speciation and evolution of local adaptation come with genomic approaches and with the development of comprehensive model systems. The marine snail Littorina saxatilis is one example of a developing marine model for investigating genetic mechanisms of rapid divergence and evolution in natural systems. This species is strongly polymorphic and shows formation of local ecotypes throughout its distribution. Support is strong for primary (in situ) and parallel formation of reproductively semi-isolated ecotypes with contact zones between heterogeneous intertidal microhabitats. This makes this species an ideal organism for gaining new insights into the interplay of divergent selection, gene flow and genetic drift during local adaptation and speciation. A relatively well-resolved draft genome and a genetic map describing 17 linkage groups (“chromosomes”) are key tools for investigating the role of structural genomic variation, such as inversions, gene duplications and translocations. Whole genome re-sequencing of pools of individuals and the first comprehensive study of a contact zone contribute direct information on selection and barriers to gene flow present in specific regions of the genome. Linking selection at the phenotypic level to patterns obser ved in the genome is under way by quantitative trait loci mapping and annotation of candidate genes, while the role of single mutations on individual fitness will have to await development of gene manipulation tools. The features of the snail system facilitate the study of local adaptation and speciation and its genomic basis, but the underlying evolutionary processes are expected to be similar in other organisms, and hence this species is a useful model.

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July 7, 2019

The state of whole-genome sequencing

Over the last decade, a technological paradigm shift has slashed the cost of DNA sequencing by over five orders of magnitude. Today, the cost of sequencing a human genome is a few thousand dollars, and it continues to fall. Here, we review the most cost-effective platforms for whole-genome sequencing (WGS) as well as emerging technologies that may displace or complement these. We also discuss the practical challenges of generating and analyzing WGS data, and how WGS has unlocked new strategies for discovering genes and variants underlying both rare and common human diseases.

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July 7, 2019

Fragmentation of surface adsorbed and aligned DNA molecules using soft lithography for next-generation sequencing

In this study, the enzymatic in situ cutting of linearized DNA molecules at approximately 11 kbp intervals is demonstrated using a soft lithography technique. The ultimate goal is to provide a general ordered cutting method to greatly simplify the assembly process. DNA was stretched onto PMMA (Poly methyl methacrylate) coated silicon by withdrawing the substrate from a DNA solution (a process termed “combing”). The stretched lambda DNA could be linearly cut with a soft lithography stamp used to selectively apply DNase I. After cutting the DNA on the substrate, the DNA fragments are removed from the surface by incubating PMMA in the commercial NEBuffer 3.1 at 75°C. The recovered fragments desorbed into the buffer and were sequenced using the PacBio RS II sequencer without an amplification step. The mean coverage was 2870X for the approximately 11 kbp fragmented sample and 100% of the lambda genome was sequenced. Methods to extend of the technique to ordered fragmentation are discussed.

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July 7, 2019

Lactobacillus allii sp. nov. isolated from scallion kimchi.

A novel strain of lactic acid bacteria, WiKim39T, was isolated from a scallion kimchi sample consisting of fermented chili peppers and vegetables. The isolate was a Gram-positive, rod-shaped, non-motile, catalase-negative and facultatively anaerobic lactic acid bacterium. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain WiKim39T belonged to the genus Lactobacillus, and shared 97.1-98.2?%?pair-wise sequence similarities with related type strains, Lactobacillus nodensis, Lactobacillus insicii, Lactobacillus versmoldensis, Lactobacillus tucceti and Lactobacillus furfuricola. The G+C?content of the strain based on its genome sequence was 35.3?mol%. The ANI values between WiKim39T and the closest relatives were lower than 80?%. Based on the phenotypic, biochemical, and phylogenetic analyses, strain WiKim39T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus allii sp. nov. is proposed. The type strain is WiKim39T (=KCTC 21077T=JCM 31938T).

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July 7, 2019

Pathogenicity and whole genome sequence analysis of a Pseudorabies virus strain FJ-2012 isolated from Fujian, Southern China.

The outbreaks of pseudorabies have been frequently reported in Bartha-K61-vaccinated farms in China since 2011. To study the pathogenicity and evolution of the circulating pseudorabies viruses in Fujian Province, mainland China, we isolated and sequenced the whole genome of a wild-type pseudorabies virus strain named “FJ-2012.” We then conducted a few downstream bioinformatics analyses including phylogenetic analysis and pathogenic analysis and used the virus to infect 6 pseudorabies virus-free piglets. FJ-2012-infected piglets developed symptoms like high body temperature and central nervous system disorders and had high mortality rate. In addition, we identified typical micropathological changes such as multiple gross lesions in infected piglets through pathological analysis and conclude that the FJ-2012 genome is significantly different from known pseudorabies viruses, in which insertions, deletions, and substitutions are observed in multiple immune and virulence genes. In summary, this study shed lights on the molecular basis of the prevalence and pathology of the pseudorabies virus strain FJ-2012. The genome of FJ-2012 could be used as a reference to study the evolution of pseudorabies viruses, which is critical to the vaccine development of new emerging pseudorabies viruses.

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July 7, 2019

Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus.

Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.

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July 7, 2019

Systems biology analysis of the key genes of surfactin production in Bacillus subtilis MJ01 (isolated from soil contaminated oil in south of Iran), spizizenii, and 168 isolates

Applying microorganism in oil recovery has attracted attentions recently. Surfactin produced by Bacillus subtilis is widely used industrially in a range of industrial applications in pharmecutical and environmental sectors. Little information about molecular mechanism of suffactin compound is available. In this study, we performed promoter and network analysis of surfactin production genes in Bacillus subtilis subsp. MJ01 (isolated from oil contaminated soil in South of Iran), spizizenii and 168. Our analysis revealed that comQ and comX are the genes with sequence alterations among these three strains of Bacillus subtilis and are involved in surfactin production. Promoter analysis indicated that lrp, argR, rpoD, purr and ihf are overrepresented and have the highest number of transcription factor binding sites (TFBs) on the key surfactin production genes in all 3 strains. Also the pattern of TFBs among these three strains was completely different. Interestingly, there is distinct difference between 168, spizizenii and MJ01 in their frequency of TFs that activate genes involve in surfactin production. Attribute weighting algorithms and decision tree analysis revealed ihf, rpoD and flHCD as the most important TF among surfactin production. Network analysis identified two significant network modules. The first one consists of key genes involved in surfactin production and the second module includes key TFs, involved in regulation of surfactin production. Our findings enhance understanding the molecular mechanism of surfactin production through systems biology analysis.

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