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Friday, July 19, 2019

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease…

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Friday, July 19, 2019

The origin of the Haitian cholera outbreak strain.

Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti.We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of…

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Friday, July 19, 2019

The complete genome sequence of Escherichia coli EC958: a high quality reference sequence for the globally disseminated multidrug resistant E. coli O25b:H4-ST131 clone.

Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific…

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Friday, July 19, 2019

New insights into dissemination and variation of the health care-associated pathogen Acinetobacter baumannii from genomic analysis.

Acinetobacter baumannii is a globally important nosocomial pathogen characterized by an increasing incidence of multidrug resistance. Routes of dissemination and gene flow among health care facilities are poorly resolved and are important for understanding the epidemiology of A. baumannii, minimizing disease transmission, and improving patient outcomes. We used whole-genome sequencing to assess diversity and genome dynamics in 49 isolates from one United States hospital system during one year from 2007 to 2008. Core single-nucleotide-variant-based phylogenetic analysis revealed multiple founder strains and multiple independent strains recovered from the same patient yet was insufficient to fully resolve strain relationships, where gene content…

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Friday, July 19, 2019

PBHoney: identifying genomic variants via long-read discordance and interrupted mapping.

As resequencing projects become more prevalent across a larger number of species, accurate variant identification will further elucidate the nature of genetic diversity and become increasingly relevant in genomic studies. However, the identification of larger genomic variants via DNA sequencing is limited by both the incomplete information provided by sequencing reads and the nature of the genome itself. Long-read sequencing technologies provide high-resolution access to structural variants often inaccessible to shorter reads.We present PBHoney, software that considers both intra-read discordance and soft-clipped tails of long reads (>10,000 bp) to identify structural variants. As a proof of concept, we identify four…

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Friday, July 19, 2019

Resolving the complexity of the human genome using single-molecule sequencing.

The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome–78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants…

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Friday, July 19, 2019

Comparative genome analysis of Wolbachia strain wAu

BACKGROUND:Wolbachia intracellular bacteria can manipulate the reproduction of their arthropod hosts, including inducing sterility between populations known as cytoplasmic incompatibility (CI). Certain strains have been identified that are unable to induce or rescue CI, including wAu from Drosophila. Genome sequencing and comparison with CI-inducing related strain wMel was undertaken in order to better understand the molecular basis of the phenotype.RESULTS:Although the genomes were broadly similar, several rearrangements were identified, particularly in the prophage regions. Many orthologous genes contained single nucleotide polymorphisms (SNPs) between the two strains, but a subset containing major differences that would likely cause inactivation in wAu were…

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Friday, July 19, 2019

Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element.

Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole-genome analysis of a methicillin-resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives…

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Friday, July 19, 2019

Genome sequencing and comparative genomics provides insights on the evolutionary dynamics and pathogenic potential of different H-serotypes of Shiga toxin-producing Escherichia coli O104.

Various H-serotypes of the Shiga toxin-producing Escherichia coli (STEC) O104, including H4, H7, H21, and H¯, have been associated with sporadic cases of illness and have caused food-borne outbreaks globally. In the U.S., STEC O104:H21 caused an outbreak associated with milk in 1994. However, there is little known on the evolutionary origins of STEC O104 strains, and how genotypic diversity contributes to pathogenic potential of various O104 H-antigen serotypes isolated from different ecological niches and/or geographical regions.Two STEC O104:H21 (milk outbreak strain) and O104:H7 (cattle isolate) strains were shot-gun sequenced, and the genomes were closed. The intimin (eae) gene, involved…

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Friday, July 19, 2019

Complete genome sequence and analysis of Lactobacillus hokkaidonensis LOOC260(T), a psychrotrophic lactic acid bacterium isolated from silage.

Lactobacillus hokkaidonensis is an obligate heterofermentative lactic acid bacterium, which is isolated from Timothy grass silage in Hokkaido, a subarctic region of Japan. This bacterium is expected to be useful as a silage starter culture in cold regions because of its remarkable psychrotolerance; it can grow at temperatures as low as 4°C. To elucidate its genetic background, particularly in relation to the source of psychrotolerance, we constructed the complete genome sequence of L. hokkaidonensis LOOC260(T) using PacBio single-molecule real-time sequencing technology.The genome of LOOC260(T) comprises one circular chromosome (2.28 Mbp) and two circular plasmids: pLOOC260-1 (81.6 kbp) and pLOOC260-2 (41.0…

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Friday, July 19, 2019

Complete nucleotide sequences of bla(CTX-M)-harboring IncF plasmids from community-associated Escherichia coli strains in the United States.

Community-associated infections due to Escherichia coli producing CTX-M-type extended-spectrum ß-lactamases are increasingly recognized in the United States. The bla(CTX-M) genes are frequently carried on IncF group plasmids. In this study, bla(CTX-M-15)-harboring plasmids pCA14 (sequence type 131 [ST131]) and pCA28 (ST44) and bla(CTX-M-14)-harboring plasmid pCA08 (ST131) were sequenced and characterized. The three plasmids were closely related to other IncFII plasmids from continents outside the United States in the conserved backbone region and multiresistance regions (MRRs). Each of the bla(CTX-M-15)-carrying plasmids pCA14 and pCA28 belonged to F31:A4:B1 (FAB [FII, FIA, FIB] formula) and showed a high level of similarity (92% coverage of…

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Friday, July 19, 2019

Assessing structural variation in a personal genome-towards a human reference diploid genome.

Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.We demonstrate Parliament’s efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation…

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Friday, July 19, 2019

Genome modification in Enterococcus faecalis OG1RF assessed by bisulfite sequencing and Single-Molecule Real-Time Sequencing.

Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged, reducing treatment options for these infections. MDR E. faecalis strains have large genomes containing mobile genetic elements (MGEs) that harbor genes for antibiotic resistance and virulence determinants. Bacteria commonly possess genome defense mechanisms to block MGE acquisition, and we hypothesize that these mechanisms have been compromised in MDR E. faecalis. In restriction-modification (R-M) defense, the bacterial genome is methylated at cytosine (C) or adenine (A) residues by a methyltransferase (MTase), such that nonself DNA can…

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Friday, July 19, 2019

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V…

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