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      Assessing the genomic relatedness and evolutionary rates of persistent verotoxigenic Escherichia coli serotypes within a closed beef herd in Canada

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          Abstract

          Verotoxigenic Escherichia coli (VTEC) are food- and water-borne pathogens associated with both sporadic illness and outbreaks of enteric disease. While it is known that cattle are reservoirs of VTEC, little is known about the genomic variation of VTEC in cattle, and whether the variation in genomes reported for human outbreak strains is consistent with individual animal or group/herd sources of infection. A previous study of VTEC prevalence identified serotypes carried persistently by three consecutive cohorts of heifers within a closed herd of cattle. This present study aimed to: (i) determine whether the genomic relatedness of bovine isolates is similar to that reported for human strains associated with single source outbreaks, (ii) estimate the rates of genome change among dominant serotypes over time within a cattle herd, and (iii) identify genomic features of serotypes associated with persistence in cattle. Illumina MiSeq genome sequencing and genotyping based on allelic and single nucleotide variations were completed, while genome change over time was measured using Bayesian evolutionary analysis sampling trees. The accessory genome, including the non-protein-encoding intergenic regions (IGRs), virulence factors, antimicrobial-resistance genes and plasmid gene content of representative persistent and sporadic cattle strains were compared using Fisher’s exact test corrected for multiple comparisons. Herd strains from serotypes O6:H34 ( n=22), O22:H8 ( n=30), O108:H8 ( n=39), O139:H19 ( n=44) and O157:H7 ( n=106) were readily distinguishable from epidemiologically unrelated strains of the same serotype using a similarity threshold of 10 or fewer allele differences between adjacent nodes. Temporal-cohort clustering within each serotype was supported by date randomization analysis. Substitutions per site per year were consistent with previously reported values for E. coli ; however, there was low branch support for these values. Acquisition of the phage-encoded Shiga toxin 2 gene in serotype O22:H8 was observed. Pan-genome analyses identified accessory regions that were more prevalent in persistent serotypes ( P≤0.05) than in sporadic serotypes. These results suggest that VTEC serotypes from a specific cattle population are highly clonal with a similar level of relatedness as human single-source outbreak-associated strains, but changes in the genome occur gradually over time. Additionally, elements in the accessory genomes may provide a selective advantage for persistence of VTEC within cattle herds.

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          FLASH: fast length adjustment of short reads to improve genome assemblies.

          Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.
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            PHYML Online—a web server for fast maximum likelihood-based phylogenetic inference

            PHYML Online is a web interface to PHYML, a software that implements a fast and accurate heuristic for estimating maximum likelihood phylogenies from DNA and protein sequences. This tool provides the user with a number of options, e.g. nonparametric bootstrap and estimation of various evolutionary parameters, in order to perform comprehensive phylogenetic analyses on large datasets in reasonable computing time. The server and its documentation are available at .
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              Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions

              Background The pan-genome of a bacterial species consists of a core and an accessory gene pool. The accessory genome is thought to be an important source of genetic variability in bacterial populations and is gained through lateral gene transfer, allowing subpopulations of bacteria to better adapt to specific niches. Low-cost and high-throughput sequencing platforms have created an exponential increase in genome sequence data and an opportunity to study the pan-genomes of many bacterial species. In this study, we describe a new online pan-genome sequence analysis program, Panseq. Results Panseq was used to identify Escherichia coli O157:H7 and E. coli K-12 genomic islands. Within a population of 60 E. coli O157:H7 strains, the existence of 65 accessory genomic regions identified by Panseq analysis was confirmed by PCR. The accessory genome and binary presence/absence data, and core genome and single nucleotide polymorphisms (SNPs) of six L. monocytogenes strains were extracted with Panseq and hierarchically clustered and visualized. The nucleotide core and binary accessory data were also used to construct maximum parsimony (MP) trees, which were compared to the MP tree generated by multi-locus sequence typing (MLST). The topology of the accessory and core trees was identical but differed from the tree produced using seven MLST loci. The Loci Selector module found the most variable and discriminatory combinations of four loci within a 100 loci set among 10 strains in 1 s, compared to the 449 s required to exhaustively search for all possible combinations; it also found the most discriminatory 20 loci from a 96 loci E. coli O157:H7 SNP dataset. Conclusion Panseq determines the core and accessory regions among a collection of genomic sequences based on user-defined parameters. It readily extracts regions unique to a genome or group of genomes, identifies SNPs within shared core genomic regions, constructs files for use in phylogeny programs based on both the presence/absence of accessory regions and SNPs within core regions and produces a graphical overview of the output. Panseq also includes a loci selector that calculates the most variable and discriminatory loci among sets of accessory loci or core gene SNPs. Availability Panseq is freely available online at http://76.70.11.198/panseq. Panseq is written in Perl.
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                Author and article information

                Journal
                Microb Genom
                Microb Genom
                mgen
                mgen
                Microbial Genomics
                Microbiology Society
                2057-5858
                June 2020
                4 June 2020
                4 June 2020
                : 6
                : 6
                : e000376
                Affiliations
                [ 1] departmentNational Microbiology Laboratory , Public Health Agency of Canada , Lethbridge, Alberta, Canada
                [ 2] Alberta Agriculture and Forestry , Lethbridge, Alberta, Canada
                [ 3] departmentNational Centre for Animal Disease , Canadian Food Inspection Agency , Lethbridge, Alberta, Canada
                [ 4] departmentNational Microbiology Laboratory , Public Health Agency of Canada , Guelph, Ontario, Canada
                Author notes
                *Correspondence: Victor P. J. Gannon, vic.gannon@ 123456canada.ca
                Author information
                https://orcid.org/0000-0001-6966-7637
                https://orcid.org/0000-0003-1162-257X
                https://orcid.org/0000-0002-5133-3409
                https://orcid.org/0000-0001-6064-405X
                Article
                000376
                10.1099/mgen.0.000376
                7371104
                32496181
                9a9df9a3-bc6d-41f0-abde-65c50d730636
                © 2020 Crown Copyright

                This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License.

                History
                : 04 October 2019
                : 20 April 2020
                Funding
                Funded by: Government of Canada Genomics and Research Development Initiative
                Award Recipient : Not Applicable
                Categories
                Research Article
                Microbial evolution and epidemiology: Population Genomics
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                escherichia coli,toxin,genomics,persistence,relatedness,evolutionary rate

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