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Asset Tag: HGAP

July 7, 2019

Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs.

Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.© 2017 Lee et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.

The human reference genome assembly plays a central role in nearly all aspects of today’s basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health. © 2017 Schneider et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

HINGE: long-read assembly achieves optimal repeat resolution.

Long-read sequencing technologies have the potential to produce gold-standard de novo genome assemblies, but fully exploiting error-prone reads to resolve repeats remains a challenge. Aggressive approaches to repeat resolution often produce misassemblies, and conservative approaches lead to unnecessary fragmentation. We present HINGE, an assembler that seeks to achieve optimal repeat resolution by distinguishing repeats that can be resolved given the data from those that cannot. This is accomplished by adding “hinges” to reads for constructing an overlap graph where only unresolvable repeats are merged. As a result, HINGE combines the error resilience of overlap-based assemblers with repeat-resolution capabilities of de Bruijn graph assemblers. HINGE was evaluated on the long-read bacterial data sets from the NCTC project. HINGE produces more finished assemblies than Miniasm and the manual pipeline of NCTC based on the HGAP assembler and Circlator. HINGE also allows us to identify 40 data sets where unresolvable repeats prevent the reliable construction of a unique finished assembly. In these cases, HINGE outputs a visually interpretable assembly graph that encodes all possible finished assemblies consistent with the reads, while other approaches such as the NCTC pipeline and FALCON either fragment the assembly or resolve the ambiguity arbitrarily.© 2017 Kamath et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Hybrid assembly of the large and highly repetitive genome of Aegilops tauschii, a progenitor of bread wheat, with the MaSuRCA mega-reads algorithm.

Long sequencing reads generated by single-molecule sequencing technology offer the possibility of dramatically improving the contiguity of genome assemblies. The biggest challenge today is that long reads have relatively high error rates, currently around 15%. The high error rates make it difficult to use this data alone, particularly with highly repetitive plant genomes. Errors in the raw data can lead to insertion or deletion errors (indels) in the consensus genome sequence, which in turn create significant problems for downstream analysis; for example, a single indel may shift the reading frame and incorrectly truncate a protein sequence. Here, we describe an algorithm that solves the high error rate problem by combining long, high-error reads with shorter but much more accurate Illumina sequencing reads, whose error rates average <1%. Our hybrid assembly algorithm combines these two types of reads to construct mega-reads, which are both long and accurate, and then assembles the mega-reads using the CABOG assembler, which was designed for long reads. We apply this technique to a large data set of Illumina and PacBio sequences from the species Aegilops tauschii, a large and extremely repetitive plant genome that has resisted previous attempts at assembly. We show that the resulting assembled contigs are far larger than in any previous assembly, with an N50 contig size of 486,807 nucleotides. We compare the contigs to independently produced optical maps to evaluate their large-scale accuracy, and to a set of high-quality bacterial artificial chromosome (BAC)-based assemblies to evaluate base-level accuracy. © 2017 Zimin et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids.

A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.

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July 7, 2019

Complex routes of nosocomial vancomycin-resistant Enterococcus faecium transmission revealed by genome sequencing.

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of nosocomial infection. Here, we describe the utility of whole-genome sequencing in defining nosocomial VREfm transmission.A retrospective study at a single hospital in the United Kingdom identified 342 patients with E. faecium bloodstream infection over 7 years. Of these, 293 patients had a stored isolate and formed the basis for the study. The first stored isolate from each case was sequenced (200 VREfm [197 vanA, 2 vanB, and 1 isolate containing both vanA and vanB], 93 vancomycin-susceptible E. faecium) and epidemiological data were collected. Genomes were also available for E. faecium associated with bloodstream infections in 15 patients in neighboring hospitals, and 456 patients across the United Kingdom and Ireland.The majority of infections in the 293 patients were hospital-acquired (n = 249) or healthcare-associated (n = 42). Phylogenetic analysis showed that 291 of 293 isolates resided in a hospital-associated clade that contained numerous discrete clusters of closely related isolates, indicative of multiple introductions into the hospital followed by clonal expansion associated with transmission. Fine-scale analysis of 6 exemplar phylogenetic clusters containing isolates from 93 patients (32%) identified complex transmission routes that spanned numerous wards and years, extending beyond the detection of conventional infection control. These contained both vancomycin-resistant and -susceptible isolates. We also identified closely related isolates from patients at Cambridge University Hospitals NHS Foundation Trust and regional and national hospitals, suggesting interhospital transmission.These findings provide important insights for infection control practice and signpost areas for interventions. We conclude that sequencing represents a powerful tool for the enhanced surveillance and control of nosocomial E. faecium transmission and infection.

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July 7, 2019

Characterization of Class IIa bacteriocin resistance in Enterococcus faecium.

Vancomycin-resistant enterococci, particularly resistant Enterococcus faecium, pose an escalating threat in nosocomial environments because of their innate resistance to many antibiotics, including vancomycin, a treatment of last resort. Many class IIa bacteriocins strongly target these enterococci and may offer a potential alternative for the management of this pathogen. However, E. faecium’s resistance to these peptides remains relatively uncharacterized. Here, we explored the development of resistance of E. faecium to a cocktail of three class IIa bacteriocins: enterocin A, enterocin P, and hiracin JM79. We started by quantifying the frequency of resistance to these peptides in four clinical isolates of E. faecium We then investigated the levels of resistance of E. faecium 6E6 mutants as well as their fitness in different carbon sources. In order to elucidate the mechanism of resistance of E. faecium to class IIa bacteriocins, we completed whole-genome sequencing of resistant mutants and performed reverse transcription-quantitative PCR (qRT-PCR) of a suspected target mannose phosphotransferase (ManPTS). We then verified this ManPTS’s role in bacteriocin susceptibility by showing that expression of the ManPTS in Lactococcus lactis results in susceptibility to the peptide cocktail. Based on the evidence found from these studies, we conclude that, in accord with other studies in E. faecalis and Listeria monocytogenes, resistance to class IIa bacteriocins in E. faecium 6E6 is likely caused by the disruption of a particular ManPTS, which we believe we have identified. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequence of Streptococcus iniae 89353, a virulent strain isolated from diseased tilapia in Taiwan.

Streptococcus iniae 89353 is a virulent strain isolated from diseased tilapia in Taiwan. The full-genome sequence of S. iniae 89353 is 2,098,647 bp. The revealed genome information will be beneficial for identification and understanding of potential virulence genes of Streptococcus iniae and possible immunogens for vaccine development against streptococcosis. Copyright © 2017 Gong et al.

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July 7, 2019

Whole-genome sequences of Burkholderia pseudomallei isolates exhibiting decreased meropenem susceptibility.

We report here paired isogenic Burkholderia pseudomallei genomes obtained from three patients receiving intravenous meropenem for melioidosis treatment, with post-meropenem isolates developing decreased susceptibility. Two genomes were finished, and four were drafted to improved high-quality standard. These genomes will be used to identify meropenem resistance mechanisms in B. pseudomallei. Copyright © 2017 Price et al.

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July 7, 2019

Isolation and genomic characterization of a Dehalococcoides strain suggests genomic rearrangement during culture.

We have developed and characterized a bacterial consortium that reductively dechlorinates trichloroethene to ethene. Quantitative PCR analysis for the 16S rRNA and reductive dehalogenase genes showed that the consortium is highly enriched with Dehalococcoides spp. that have two vinyl chloride reductive dehalogenase genes, bvcA and vcrA, and a trichloroethene reductive dehalogenase gene, tceA. The metagenome analysis of the consortium by the next generation sequencer SOLiD 3 Plus suggests that a Dehalococcoides sp. that is highly homologous to D. mccartyi 195 and equipped with vcrA and tceA exists in the consortium. We isolated this Dehalococcoides sp. and designated it as D. mccartyi UCH-ATV1. As the growth of D. mccartyi UCH-ATV1 is too slow under isolated conditions, we constructed a consortium by mixing D. mccartyi UCH-ATV1 with several other bacteria and performed metagenomic sequencing using the single molecule DNA sequencer PacBio RS II. We successfully determined the complete genome sequence of D. mccartyi UCH-ATV1. The strain is equipped with vcrA and tceA, but lacks bvcA. Comparison with tag sequences of SOLiD 3 Plus from the original consortium shows a few differences between the sequences. This suggests that a genome rearrangement of Dehalococcoides sp. occurred during culture.

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