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July 7, 2019

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing.

The PacBio RS II provides for single molecule, real-time DNA technology to sequence genomes and detect DNA modifications. The starting point for high-quality sequence production is high molecular weight genomic DNA. To automate the library preparation process, there must be high-throughput methods in place to assess the genomic DNA, to ensure the size and amounts of the sheared DNA fragments and final library.The library construction automation was accomplished using the Agilent NGS workstation with Bravo accessories for heating, shaking, cooling, and magnetic bead manipulations for template purification. The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated.Automated protocols of PacBio 10 kb library preparation produced libraries with similar technical performance to those generated manually. The TapeStation System proved to be a reliable method that could be used in a 96-well plate format to QC the DNA equivalent to the standard Bioanalyzer System results. The DNA Integrity Number that is calculated in the TapeStation System software upon analysis of genomic DNA is quite helpful to assure that the starting genomic DNA is not degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.


July 7, 2019

Complete genome sequence of Acetobacter pomorum Oregon-R-modENCODE strain BDGP5, an acetic acid bacterium found in the Drosophila melanogaster gut.

Acetobacter pomorum Oregon-R-modENCODE strain BDGP5 was isolated from Drosophila melanogaster for functional host-microbe interaction studies. The complete genome is composed of a single chromosomal circle of 2,848,089 bp, with a G+C content of 53% and three plasmids of 131,455 bp, 19,216 bp, and 9,160 bp. Copyright © 2017 Wan et al.


July 7, 2019

Closed genome sequence of Chryseobacterium piperi strain CTMT/ATCC BAA-1782, a Gram-negative bacterium with clostridial neurotoxin-like coding sequences.

Clostridial neurotoxins, including botulinum and tetanus neurotoxins, are among the deadliest known bacterial toxins. Until recently, the horizontal mobility of this toxin gene family appeared to be limited to the genusClostridiumWe report here the closed genome sequence ofChryseobacterium piperi, a Gram-negative bacterium containing coding sequences with homology to clostridial neurotoxin family proteins. Copyright © 2017 Wentz et al.


July 7, 2019

Complete genome sequence of Bacillus velezensis L-1, which has antagonistic activity against pear diseases.

Bacillus velezensis L-1 is an effective biocontrol agent against pear diseases. Here, we report the complete genome sequence of B. velezensis L-1 in which clusters related to the biosynthesis of secondary metabolites were predicted. This genome provides insights into the possible biocontrol mechanisms and furthers application of this specific bacterium. Copyright © 2017 Sun et al.


July 7, 2019

Characterization of Fusobacterium varium Fv113-g1 isolated from a patient with ulcerative colitis based on complete genome sequence and transcriptome analysis.

Fusobacterium spp. present in the oral and gut flora is carcinogenic and is associated with the risk of pancreatic and colorectal cancers. Fusobacterium spp. is also implicated in a broad spectrum of human pathologies, including Crohn’s disease and ulcerative colitis (UC). Here we report the complete genome sequence of Fusobacterium varium Fv113-g1 (genome size, 3.96 Mb) isolated from a patient with UC. Comparative genome analyses totally suggested that Fv113-g1 is basically assigned as F. varium, in particular, it could be reclassified as notable F. varium subsp. similar to F. ulcerans because of partial shared orthologs. Compared with the genome sequences of F. varium ATCC 27725 (genome size, 3.30 Mb) and other strains of Fusobacterium spp., Fv113-g1 possesses many accessary pan-genome sequences with noteworthy multiple virulence factors, including 44 autotransporters (type V secretion system, T5SS) and 13 Fusobacterium adhesion (FadA) paralogs involved in potential mucosal inflammation. Indeed, transcriptome analysis demonstrated that Fv113-g1-specific accessary genes, such as multiple T5SS and fadA paralogs, showed notably increased expression with D-MEM cultivation than with brain heart infusion broth. This implied that growth condition may enhance the expression of such potential virulence factors, leading to remarkable survival against other gut microorganisms and to the pathogenicity to human intestinal epithelium.


July 7, 2019

A 3-way hybrid approach to generate a new high-quality chimpanzee reference genome (Pan_tro_3.0).

The chimpanzee is arguably the most important species for the study of human origins. A key resource for these studies is a high-quality reference genome assembly; however, as with most mammalian genomes, the current iteration of the chimpanzee reference genome assembly is highly fragmented. In the current iteration of the chimpanzee reference genome assembly (Pan_tro_2.1.4), the sequence is scattered across more then 183 000 contigs, incorporating more than 159 000 gaps, with a genome-wide contig N50 of 51 Kbp. In this work, we produce an extensive and diverse array of sequencing datasets to rapidly assemble a new chimpanzee reference that surpasses previous iterations in bases represented and organized in large scaffolds. To this end, we show substantial improvements over the current release of the chimpanzee genome (Pan_tro_2.1.4) by several metrics, such as increased contiguity by >750% and 300% on contigs and scaffolds, respectively, and closure of 77% of gaps in the Pan_tro_2.1.4 assembly gaps spanning >850 Kbp of the novel coding sequence based on RNASeq data. We further report more than 2700 genes that had putatively erroneous frame-shift predictions to human in Pan_tro_2.1.4 and show a substantial increase in the annotation of repetitive elements. We apply a simple 3-way hybrid approach to considerably improve the reference genome assembly for the chimpanzee, providing a valuable resource for the study of human origins. Furthermore, we produce extensive sequencing datasets that are all derived from the same cell line, generating a broad non-human benchmark dataset.© The Author 2017. Published by Oxford University Press.


July 7, 2019

Trajectories and drivers of genome evolution in surface-associated marine Phaeobacter.

The extent of genome divergence and the evolutionary events leading to speciation of marine bacteria have mostly been studied for (locally) abundant, free-living groups. The genus Phaeobacter is found on different marine surfaces, seems to occupy geographically disjunct habitats, and is involved in different biotic interactions, and was therefore targeted in the present study. The analysis of the chromosomes of 32 closely related but geographically spread Phaeobacter strains revealed an exceptionally large, highly syntenic core genome. The flexible gene pool is constantly but slightly expanding across all Phaeobacter lineages. The horizontally transferred genes mostly originated from bacteria of the Roseobacter group and horizontal transfer most likely was mediated by gene transfer agents. No evidence for geographic isolation and habitat specificity of the different phylogenomic Phaeobacter clades was detected based on the sources of isolation. In contrast, the functional gene repertoire and physiological traits of different phylogenomic Phaeobacter clades were sufficiently distinct to suggest an adaptation to an associated lifestyle with algae, to additional nutrient sources, or toxic heavy metals. Our study reveals that the evolutionary trajectories of surface-associated marine bacteria can differ significantly from free-living marine bacteria or marine generalists.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


July 7, 2019

Complete genome sequence of Bacillus altitudinis P-10, a potential bioprotectant against Xanthomonas oryzae pv. oryzae, isolated from rice rhizosphere in Java, Indonesia.

Bacillus altitudinis P-10 was isolated from the rhizosphere of rice grown in an organic rice field and provides strong antagonism against the bacterial blight caused by Xanthomonas oryzae pv. oryzae in rice. Herein, we provide the complete genome sequence and a possible explanation of the antibiotic function of the P-10 strain.


July 7, 2019

Sex-specific influences of mtDNA mitotype and diet on mitochondrial functions and physiological traits in Drosophila melanogaster.

Here we determine the sex-specific influence of mtDNA type (mitotype) and diet on mitochondrial functions and physiology in two Drosophila melanogaster lines. In many species, males and females differ in aspects of their energy production. These sex-specific influences may be caused by differences in evolutionary history and physiological functions. We predicted the influence of mtDNA mutations should be stronger in males than females as a result of the organelle’s maternal mode of inheritance in the majority of metazoans. In contrast, we predicted the influence of diet would be greater in females due to higher metabolic flexibility. We included four diets that differed in their protein: carbohydrate (P:C) ratios as they are the two-major energy-yielding macronutrients in the fly diet. We assayed four mitochondrial function traits (Complex I oxidative phosphorylation, reactive oxygen species production, superoxide dismutase activity, and mtDNA copy number) and four physiological traits (fecundity, longevity, lipid content, and starvation resistance). Traits were assayed at 11 d and 25 d of age. Consistent with predictions we observe that the mitotype influenced males more than females supporting the hypothesis of a sex-specific selective sieve in the mitochondrial genome caused by the maternal inheritance of mitochondria. Also, consistent with predictions, we found that the diet influenced females more than males.


July 7, 2019

De novo design and synthesis of a 30-cistron translation-factor module.

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.


July 7, 2019

Nitrogen fixation genes and nitrogenase activity of the non-heterocystous cyanobacterium Thermoleptolyngbya sp. O-77.

Cyanobacteria are widely distributed in marine, aquatic, and terrestrial ecosystems, and play an important role in the global nitrogen cycle. In the present study, we examined the genome sequence of the thermophilic non-heterocystous N2-fixing cyanobacterium, Thermoleptolyngbya sp. O-77 (formerly known as Leptolyngbya sp. O-77) and characterized its nitrogenase activity. The genome of this cyanobacterial strain O-77 consists of a single chromosome containing a nitrogen fixation gene cluster. A phylogenetic analysis indicated that the NifH amino acid sequence from strain O-77 was clustered with those from a group of mesophilic species: the highest identity was found in Leptolyngbya sp. KIOST-1 (97.9% sequence identity). The nitrogenase activity of O-77 cells was dependent on illumination, whereas a high intensity of light of 40 µmol m-2 s-1 suppressed the effects of illumination.


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