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Asset Tag: Third generation sequencing

July 7, 2019

Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.

DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.

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July 7, 2019

Complete genome sequence of a multidrug-resistant Salmonella enterica serovar Typhimurium var. 5- strain isolated from chicken breast.

Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of salmonellosis. Here, we report a closed genome sequence, including sequences of 3 plasmids, of Salmonella serovar Typhimurium var. 5- CFSAN001921 (National Antimicrobial Resistance Monitoring System [NARMS] strain ID N30688), which was isolated from chicken breast meat and shows resistance to 10 different antimicrobials. Whole-genome and plasmid sequence analyses of this isolate will help enhance our understanding of this pathogenic multidrug-resistant serovar.

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July 7, 2019

Genome sequence of Phaeobacter daeponensis type strain (DSM 23529(T)), a facultatively anaerobic bacterium isolated from marine sediment, and emendation of Phaeobacter daeponensis.

TF-218(T) is the type strain of the species Phaeobacter daeponensis Yoon et al. 2007, a facultatively anaerobic Phaeobacter species isolated from tidal flats. Here we describe the draft genome sequence and annotation of this bacterium together with previously unreported aspects of its phenotype. We analyzed the genome for genes involved in secondary metabolite production and its anaerobic lifestyle, which have also been described for its closest relative Phaeobacter caeruleus. The 4,642,596 bp long genome of strain TF-218(T) contains 4,310 protein-coding genes and 78 RNA genes including four rRNA operons and consists of five replicons: one chromosome and four extrachromosomal elements with sizes of 276 kb, 174 kb, 117 kb and 90 kb. Genome analysis showed that TF-218(T) possesses all of the genes for indigoidine biosynthesis, and on specific media the strain showed a blue pigmentation. We also found genes for dissimilatory nitrate reduction, gene-transfer agents, NRPS/ PKS genes and signaling systems homologous to the LuxR/I system.

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July 7, 2019

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

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July 7, 2019

Precise breakpoint localization of large genomic deletions using PacBio and Illumina next-generation sequencers.

Herein we present the applicability of single-molecule (PacBio RS) and second-generation sequencing technology (Illumina) to the characterization of large genomic deletions. By testing samples previously characterized using a Sanger approach, our methods determined that both next-generation sequencing platforms were able to identify the position of deletion breakpoints. Our results point out various advantages of next-generation sequencing platforms when characterizing genomic deletions; however, special attention must be dedicated to identical sequences flanking the breakpoints, such as poly(N) motifs.

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July 7, 2019

Complete genome sequence of Staphylococcus aureus Z172, a vancomycin-intermediate and daptomycin-nonsusceptible methicillin-resistant strain isolated in Taiwan.

We report the complete genome sequence of Z172, a representative strain of sequence type 239-staphylococcal cassette chromosome mec type III (ST239-SCCmec type III) hospital-associated methicillin-resistant Staphylococcus aureus in Taiwan. Strain Z172 also exhibits a vancomycin-intermediate and daptomycin-nonsusceptible phenotype.

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July 7, 2019

PBSIM: PacBio reads simulator–toward accurate genome assembly.

PacBio sequencers produce two types of characteristic reads (continuous long reads: long and high error rate and circular consensus sequencing: short and low error rate), both of which could be useful for de novo assembly of genomes. Currently, there is no available simulator that targets the specific generation of PacBio libraries.Our analysis of 13 PacBio datasets showed characteristic features of PacBio reads (e.g. the read length of PacBio reads follows a log-normal distribution). We have developed a read simulator, PBSIM, that captures these features using either a model-based or sampling-based method. Using PBSIM, we conducted several hybrid error correction and assembly tests for PacBio reads, suggesting that a continuous long reads coverage depth of at least 15 in combination with a circular consensus sequencing coverage depth of at least 30 achieved extensive assembly results.PBSIM is freely available from the web under the GNU GPL v2 license (http://code.google.com/p/pbsim/).

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July 7, 2019

Complete genome sequence of Leifsonia xyli subsp. cynodontis strain DSM46306, a gram-positive bacterial pathogen of grasses.

We announce the complete genome sequence of Leifsonia xyli subsp. cynodontis, a vascular pathogen of Bermuda grass. The species also comprises Leifsonia xyli subsp. xyli, a sugarcane pathogen. Since these two subspecies have genome sequences available, a comparative analysis will contribute to our understanding of the differences in their biology and host specificity.

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July 7, 2019

Draft genome of Spiribacter salinus M19-40, an abundant gammaproteobacterium in aquatic hypersaline environments.

We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.

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July 7, 2019

Mutation in the C-di-AMP cyclase dacA affects fitness and resistance of methicillin resistant Staphylococcus aureus.

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.

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July 7, 2019

The impact of aminoglycosides on the dynamics of translation elongation.

Inferring antibiotic mechanisms on translation through static structures has been challenging, as biological systems are highly dynamic. Dynamic single-molecule methods are also limited to few simultaneously measurable parameters. We have circumvented these limitations with a multifaceted approach to investigate three structurally distinct aminoglycosides that bind to the aminoacyl-transfer RNA site (A site) in the prokaryotic 30S ribosomal subunit: apramycin, paromomycin, and gentamicin. Using several single-molecule fluorescence measurements combined with structural and biochemical techniques, we observed distinct changes to translational dynamics for each aminoglycoside. While all three drugs effectively inhibit translation elongation, their actions are structurally and mechanistically distinct. Apramycin does not displace A1492 and A1493 at the decoding center, as demonstrated by a solution nuclear magnetic resonance structure, causing only limited miscoding; instead, it primarily blocks translocation. Paromomycin and gentamicin, which displace A1492 and A1493, cause significant miscoding, block intersubunit rotation, and inhibit translocation. Our results show the power of combined dynamics, structural, and biochemical approaches to elucidate the complex mechanisms underlying translation and its inhibition. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

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