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Auto Tag: Base modification

July 7, 2019

Comparative genomics of the Campylobacter lari group.

The Campylobacter lari group is a phylogenetic clade within the epsilon subdivision of the Proteobacteria and is part of the thermotolerant Campylobacter spp., a division within the genus that includes the human pathogen Campylobacter jejuni. The C. lari group is currently composed of five species (C. lari, Campylobacter insulaenigrae, Campylobacter volucris, Campylobacter subantarcticus, and Campylobacter peloridis), as well as a group of strains termed the urease-positive thermophilic Campylobacter (UPTC) and other C. lari-like strains. Here we present the complete genome sequences of 11 C. lari group strains, including the five C. lari group species, four UPTC strains, and a lari-like strain isolated in this study. The genome of C. lari subsp. lari strain RM2100 was described previously. Analysis of the C. lari group genomes indicates that this group is highly related at the genome level. Furthermore, these genomes are strongly syntenic with minor rearrangements occurring only in 4 of the 12 genomes studied. The C. lari group can be bifurcated, based on the flagella and flagellar modification genes. Genomic analysis of the UPTC strains indicated that these organisms are variable but highly similar, closely related to but distinct from C. lari. Additionally, the C. lari group contains multiple genes encoding hemagglutination domain proteins, which are either contingency genes or linked to conserved contingency genes. Many of the features identified in strain RM2100, such as major deficiencies in amino acid biosynthesis and energy metabolism, are conserved across all 12 genomes, suggesting that these common features may play a role in the association of the C. lari group with coastal environments and watersheds. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2014. This work is written by US Government employees and is in the public domain in the US.

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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

Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation.

We have developed a sequencing method on the Pacific Biosciences RS sequencer (the PacBio) for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400-500 ng of sheared DNA for the library preparation.

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July 7, 2019

Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine.

We describe strand-specific, base-resolution detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA with single-molecule sensitivity, combining a bioorthogonal, selective chemical labeling method of 5-hmC with single-molecule, real-time (SMRT) DNA sequencing. The chemical labeling not only allows affinity enrichment of 5-hmC-containing DNA fragments but also enhances the kinetic signal of 5-hmC during SMRT sequencing. We applied the approach to sequence 5-hmC in a genomic DNA sample with high confidence.

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July 7, 2019

Direct detection and sequencing of damaged DNA bases.

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template – as a by-product of the sequencing method – through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

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July 7, 2019

Implementation and data analysis of Tn-seq, whole genome resequencing, and single-molecule real time sequencing for bacterial genetics.

Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remains a challenge for many. In this minireview, we have chosen to summarize three sequencing approaches that are particularly useful for bacterial genetics. We provide resources for scientists new to and interested in their application. Herein, we discuss the analysis of Tn-seq data to determine gene disruptions differentially represented in a mutant population, Illumina sequencing for identification of suppressor or other mutations, and we summarize single-molecule real time (SMRT) sequencing for de novo genome assembly and the use of the output data for detection of DNA base modifications. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Evolutionary origins of the emergent ST796 clone of vancomycin resistant Enterococcus faecium.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

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July 7, 2019

GenBank.

GenBank(®) (www.ncbi.nlm.nih.gov/genbank/) is a comprehensive database that contains publicly available nucleotide sequences for 370 000 formally described species. These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or the NCBI Submission Portal. GenBank staff assign accession numbers upon data receipt. Daily data exchange with the European Nucleotide Archive (ENA) and the DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through the NCBI Nucleotide database, which links to related information such as taxonomy, genomes, protein sequences and structures, and biomedical journal literature in PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. Recent updates include changes to policies regarding sequence identifiers, an improved 16S submission wizard, targeted loci studies, the ability to submit methylation and BioNano mapping files, and a database of anti-microbial resistance genes. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019

Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of loci encoding increased protein quality control mechanisms.

Salmonella enterica subsp. enterica bacteria are important foodborne pathogens with major economic impact. Some isolates exhibit increased heat tolerance, a concern for food safety. Analysis of a finished-quality genome sequence of an isolate commonly used in heat resistance studies, S. enterica subsp. enterica serovar Senftenberg 775W (ATCC 43845), demonstrated an interesting observation that this strain contains not just one, but two horizontally acquired thermotolerance locus homologs. These two loci reside on a large 341.3-kbp plasmid that is similar to the well-studied IncHI2 R478 plasmid but lacks any antibiotic resistance genes found on R478 or other IncHI2 plasmids. As this historical Salmonella isolate has been in use since 1941, comparative analysis of the plasmid and of the thermotolerance loci contained on the plasmid will provide insight into the evolution of heat resistance loci as well as acquisition of resistance determinants in IncHI2 plasmids. IMPORTANCE Thermal interventions are commonly used in the food industry as a means of mitigating pathogen contamination in food products. Concern over heat-resistant food contaminants has recently increased, with the identification of a conserved locus shown to confer heat resistance in disparate lineages of Gram-negative bacteria. Complete sequence analysis of a historical isolate of Salmonella enterica serovar Senftenberg, used in numerous studies because of its novel heat resistance, revealed that this important strain possesses two distinct copies of this conserved thermotolerance locus, residing on a multireplicon IncHI2/IncHI2A plasmid. Phylogenetic analysis of these loci in comparison with homologs identified in various bacterial genera provides an opportunity to examine the evolution and distribution of loci conferring resistance to environmental stressors, such as heat and desiccation.

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July 7, 2019

Free-living Enterobacterium Pragia fontium 24613: complete genome sequence and metabolic profiling.

Pragia fontium is one of the few species that belongs to the group of atypical hydrogen sulfide-producing enterobacteria. Unlike other members of this closely related group, P. fontium is not associated with any known host and has been reported as a free-living bacterium. Whole genome sequencing and metabolic fingerprinting confirmed the phylogenetic position of P. fontium inside the group of atypical H2S producers. Genomic data have revealed that P. fontium 24613 has limited pathogenic potential, although there are signs of genome decay. Although the lack of specific virulence factors and no association with a host species suggest a free-living style, the signs of genome decay suggest a process of adaptation to an as-yet-unknown host.

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July 7, 2019

Genomics-enabled analysis of the emergent disease cotton bacterial blight.

Cotton bacterial blight (CBB), an important disease of (Gossypium hirsutum) in the early 20th century, had been controlled by resistant germplasm for over half a century. Recently, CBB re-emerged as an agronomic problem in the United States. Here, we report analysis of cotton variety planting statistics that indicate a steady increase in the percentage of susceptible cotton varieties grown each year since 2009. Phylogenetic analysis revealed that strains from the current outbreak cluster with race 18 Xanthomonas citri pv. malvacearum (Xcm) strains. Illumina based draft genomes were generated for thirteen Xcm isolates and analyzed along with 4 previously published Xcm genomes. These genomes encode 24 conserved and nine variable type three effectors. Strains in the race 18 clade contain 3 to 5 more effectors than other Xcm strains. SMRT sequencing of two geographically and temporally diverse strains of Xcm yielded circular chromosomes and accompanying plasmids. These genomes encode eight and thirteen distinct transcription activator-like effector genes. RNA-sequencing revealed 52 genes induced within two cotton cultivars by both tested Xcm strains. This gene list includes a homeologous pair of genes, with homology to the known susceptibility gene, MLO. In contrast, the two strains of Xcm induce different clade III SWEET sugar transporters. Subsequent genome wide analysis revealed patterns in the overall expression of homeologous gene pairs in cotton after inoculation by Xcm. These data reveal important insights into the Xcm-G. hirsutum disease complex and strategies for future development of resistant cultivars.

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July 7, 2019

SMRT Sequencing revealed mitogenome characteristics and mitogenome-wide DNA modification pattern in Ophiocordyceps sinensis.

Single molecule, real-time (SMRT) sequencing was used to characterize mitochondrial (mt) genome of Ophiocordyceps sinensis and to analyze the mt genome-wide pattern of epigenetic DNA modification. The complete mt genome of O. sinensis, with a size of 157,539 bp, is the fourth largest Ascomycota mt genome sequenced to date. It contained 14 conserved protein-coding genes (PCGs), 1 intronic protein rps3, 27 tRNAs and 2 rRNA subunits, which are common characteristics of the known mt genomes in Hypocreales. A phylogenetic tree inferred from 14 PCGs in Pezizomycotina fungi supports O. sinensis as most closely related to Hirsutella rhossiliensis in Ophiocordycipitaceae. A total of 36 sequence sites in rps3 were under positive selection, with dN/dS >1 in the 20 compared fungi. Among them, 16 sites were statistically significant. In addition, the mt genome-wide base modification pattern of O. sinensis was determined in this study, especially DNA methylation. The methylations were located in coding and uncoding regions of mt PCGs in O. sinensis, and might be closely related to the expression of PCGs or the binding affinity of transcription factor A to mtDNA. Consequently, these methylations may affect the enzymatic activity of oxidative phosphorylation and then the mt respiratory rate; or they may influence mt biogenesis. Therefore, methylations in the mitogenome of O. sinensis might be a genetic feature to adapt to the cold and low PO2 environment at high altitude, where O. sinensis is endemic. This is the first report on epigenetic modifications in a fungal mt genome.

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July 7, 2019

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism’s physiology, metabolism, and pathogenesis in human disease.

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July 7, 2019

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing.

Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides-zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000-base-pair-long DNA template within seconds from a sub-nanogram input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

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