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Asset Tag: Microbiology

July 19, 2019

Returning to more finished genomes

Abstract Genomic data have become commonplace in most branches of the biological sciences and have fundamentally altered the way research is conducted. However, the predominance of short-read sequence data from second-generation sequencing technologies has commonly resulted in fragmented and partial genomic data characteristics. In this opinion, I will highlight how long, unbiased reads from single molecule, real-time (SMRT) sequencing now allow for a return to more contiguous and comprehensive views of genomes.

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July 19, 2019

The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development.

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement. Copyright © 2014 Elsevier Inc. All rights reserved.

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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 structural variants and two genomic features in a strain of Escherichia coli with PBHoney and validate them via de novo assembly. PBHoney is available for download at http://sourceforge.net/projects/pb-jelly/.Implementing two variant-identification approaches that exploit the high mappability of long reads, PBHoney is demonstrated as being effective at detecting larger structural variants using whole-genome Pacific Biosciences RS II Continuous Long Reads. Furthermore, PBHoney is able to discover two genomic features: the existence of Rac-Phage in isolate; evidence of E. coli’s circular genome.

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July 19, 2019

Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis.

More than 20% of the world’s population is at risk for infection by filarial nematodes and >180 million people worldwide are already infected. Along with infection comes significant morbidity that has a socioeconomic impact. The eight filarial nematodes that infect humans are Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi, of which three have published draft genome sequences. Since all have humans as the definitive host, standard avenues of research that rely on culturing and genetics have often not been possible. Therefore, genome sequencing provides an important window into understanding the biology of these parasites. The need for large amounts of high quality genomic DNA from homozygous, inbred lines; the availability of only short sequence reads from next-generation sequencing platforms at a reasonable expense; and the lack of random large insert libraries has limited our ability to generate high quality genome sequences for these parasites. However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries.Here, we report on efforts to generate a more complete genome assembly for L. loa using genetically heterogeneous DNA isolated from a single clinical sample and sequenced on the Pacific Biosciences platform. To obtain the best assembly, numerous assemblers and sequencing datasets were analyzed, combined, and compared. Quiver-informed trimming of an assembly of only Pacific Biosciences reads by HGAP2 was selected as the final assembly of 96.4 Mbp in 2,250 contigs. This results in ~9% more of the genome in ~85% fewer contigs from ~80% less starting material at a fraction of the cost of previous Roche 454-based sequencing efforts.The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.

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July 19, 2019

Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1.

We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication.

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July 19, 2019

Entering the era of bacterial epigenomics with single molecule real time DNA sequencing.

DNA modifications, such as methylation guide numerous critical biological processes, yet epigenetic information has not routinely been collected as part of DNA sequence analyses. Recently, the development of single molecule real time (SMRT) DNA sequencing has enabled detection of modified nucleotides (e.g. 6mA, 4mC, 5mC) in parallel with acquisition of primary sequence data, based on analysis of the kinetics of DNA synthesis reactions. In bacteria, genome-wide mapping of methylated and unmethylated loci is now feasible. This technological advance sets the stage for comprehensive, mechanistic assessment of the effects of bacterial DNA methyltransferases (MTases)-which are ubiquitous, extremely diverse, and largely uncharacterized-on gene expression, chromosome structure, chromosome replication, and other fundamental biological processes. SMRT sequencing also enables detection of damaged DNA and has the potential to uncover novel DNA modifications. Copyright © 2013 Elsevier Ltd. All rights reserved.

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July 19, 2019

Full-length haplotype reconstruction to infer the structure of heterogeneous virus populations.

Next-generation sequencing (NGS) technologies enable new insights into the diversity of virus populations within their hosts. Diversity estimation is currently restricted to single-nucleotide variants or to local fragments of no more than a few hundred nucleotides defined by the length of sequence reads. To study complex heterogeneous virus populations comprehensively, novel methods are required that allow for complete reconstruction of the individual viral haplotypes. Here, we show that assembly of whole viral genomes of ~8600 nucleotides length is feasible from mixtures of heterogeneous HIV-1 strains derived from defined combinations of cloned virus strains and from clinical samples of an HIV-1 superinfected individual. Haplotype reconstruction was achieved using optimized experimental protocols and computational methods for amplification, sequencing and assembly. We comparatively assessed the performance of the three NGS platforms 454 Life Sciences/Roche, Illumina and Pacific Biosciences for this task. Our results prove and delineate the feasibility of NGS-based full-length viral haplotype reconstruction and provide new tools for studying evolution and pathogenesis of viruses.© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

Comparative genomic analysis and virulence differences in closely related Salmonella enterica serotype Heidelberg isolates from humans, retail meats, and animals.

Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. Recently, an antibiotic-resistant strain of this serovar was implicated in a large 2011 multistate outbreak resulting from consumption of contaminated ground turkey that involved 136 confirmed cases, with one death. In this study, we assessed the evolutionary diversity of 44 S. Heidelberg isolates using whole-genome sequencing (WGS) generated by the 454 GS FLX (Roche) platform. The isolates, including 30 with nearly indistinguishable (one band difference) Xbal pulsed-field gel electrophoresis patterns (JF6X01.0032, JF6X01.0058), were collected from various sources between 1982 and 2011 and included nine isolates associated with the 2011 outbreak. Additionally, we determined the complete sequence for the chromosome and three plasmids from a clinical isolate associated with the 2011 outbreak using the Pacific Biosciences (PacBio) system. Using single-nucleotide polymorphism (SNP) analyses, we were able to distinguish highly clonal isolates, including strains isolated at different times in the same year. The isolates from the recent 2011 outbreak clustered together with a mean SNP variation of only 17 SNPs. The S. Heidelberg isolates carried a variety of phages, such as prophage P22, P4, lambda-like prophage Gifsy-2, and the P2-like phage which carries the sopE1 gene, virulence genes including 62 pathogenicity, and 13 fimbrial markers and resistance plasmids of the incompatibility (Inc)I1, IncA/C, and IncHI2 groups. Twenty-one strains contained an IncX plasmid carrying a type IV secretion system. On the basis of the recent and historical isolates used in this study, our results demonstrated that, in addition to providing detailed genetic information for the isolates, WGS can identify SNP targets that can be utilized for differentiating highly clonal S. Heidelberg isolates.

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July 19, 2019

Evolutionary dynamics of Vibrio cholerae O1 following a single-source introduction to Haiti.

Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics.Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.

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July 19, 2019

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome.After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51?N-6-methyladenines and 152?N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5a as a host bacterium to construct a shotgun library.This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.

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July 19, 2019

Recently published Streptomyces genome sequences.

Many readers of this journal will need no introduction to the bacterial genus Streptomyces, which includes several hundred species, many of which produce biotechnologically useful secondary metabolites. The last 2 years have seen numerous publications describing Streptomyces genome sequences (Table?1), mostly as short genome announcements restricted to just 500 words and therefore allowing little description and analysis. Our aim in this current manuscript is to survey these recent publications and to dig a little deeper where appropriate. The genus Streptomyces is now one of the most highly sequenced, with 19 finished genomic sequences (Table?2) and a further 125 draft assemblies available in the GenBank database as of 3rd of May 2014; by the time this is published, no doubt there will be more. The reasons given for sequencing this latest crop of Streptomyces include production of industrially important enzymes, degradation of lignin, antibiotic production, rapid growth and halo-tolerance and an endophytic lifestyle (Table?1).

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July 19, 2019

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.

The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.

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July 19, 2019

Reducing assembly complexity of microbial genomes with single-molecule sequencing.

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem.To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

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July 19, 2019

Inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses.

No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2′-C-methylcytidine (2′-CMC), and interferon alpha 2a (IFN-a2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-a2a and 2′-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-a2a and 6-azaU was moderately synergistic. The combination of 2′-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2′-CMC, AHFV variants with reduced susceptibility to 2′-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2′-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2′-CMC. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

Population structure of KPC-producing Klebsiella pneumoniae isolates from midwestern U.S. hospitals.

Genome sequencing of carbapenem-resistant Klebsiella pneumoniae isolates from regional U.S. hospitals was used to characterize strain diversity and the bla(KPC) genetic context. A phylogeny based on core single-nucleotide variants (SNVs) supports a division of sequence type 258 (ST258) into two distinct groups. The primary differences between the groups are in the capsular polysaccharide locus (cps) and their plasmid contents. A strict association between clade and KPC variant was found. The bla(KPC) gene was found on variants of two plasmid backbones. This study indicates that highly similar K. pneumoniae subpopulations coexist within the same hospitals over time. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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