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High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach.

Allard MW, Luo Y, Strain E, Li C, Keys CE, Son I, Stones R, Musser SM, Brown EW - BMC Genomics (2012)

Bottom Line: In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data.Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications.This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Regulatory Science, Center for Food Safety & Applied Nutrition, U,S, Food & Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740, USA. Marc.Allard@fda.hhs.gov

ABSTRACT

Background: Next-Generation Sequencing (NGS) is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study.

Results: Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data.

Conclusions: Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications. When draft genomes are collected at 15×-20× coverage and passed through a quality filter as part of a data analysis pipeline, including sub-passaged replicates defined by a few SNPs, they can be accurately placed in a phylogenetic context. This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.

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Phylogenetic diversity and relationships among a single S. Montevideo clone. GARLI phylogenetic analysis of the outbreak isolates was performed on a set of 43 concatenated ORFs containing informative SNPs (Table 3). Terminal names, scale bar, branch lengths and bootstrap scores are as in Figure 1. Numbers above the branches represent unique SNPs that define these internal branches. The phylogenetic analysis reported here partitions the S. Montevideo clone into 6 lineages (A-F) and expands upon a previous tree [5] with the inclusion of 5 more strains and the noted expansion of outbreak strains into clade E. To the right of the tree, each isolate is labeled with the Not1 pattern that was determined using PFGE with each unique number identifying a new Not1 pattern.
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Figure 3: Phylogenetic diversity and relationships among a single S. Montevideo clone. GARLI phylogenetic analysis of the outbreak isolates was performed on a set of 43 concatenated ORFs containing informative SNPs (Table 3). Terminal names, scale bar, branch lengths and bootstrap scores are as in Figure 1. Numbers above the branches represent unique SNPs that define these internal branches. The phylogenetic analysis reported here partitions the S. Montevideo clone into 6 lineages (A-F) and expands upon a previous tree [5] with the inclusion of 5 more strains and the noted expansion of outbreak strains into clade E. To the right of the tree, each isolate is labeled with the Not1 pattern that was determined using PFGE with each unique number identifying a new Not1 pattern.

Mentions: The importance of NGS in ascertaining high-resolution phylogenetic and molecular epidemiological histories of infectious outbreak clones of bacterial pathogens has recently been noted [18,20]. In the current study, NGS was applied for reconstructing the detailed evolutionary genetic structure of an individual clone of S. Montevideo that is largely indistinguishable using PFGE. Specifically, NGS analysis was applied to a set of S. Montevideo isolates either associated with or genetically homologous to a food contamination event of spiced Italian-style meats in the U.S. in 2009 and 2010 http://www.cdc.gov/Salmonella/montevideo/index.html. We reported previously on the success of NGS for distinguishing some of these isolates from other clonally related isolates unassociated with this spiced-meat S. Montevideo outbreak [5]. Herein, we combined the genomes of 34 highly homogeneous S. Montevideos from food, environmental, and clinical sources from this spiced-meat outbreak with 24 newly sequenced (~15X) S. Montevideo genomes derived from clinical-food matches associated with the same spiced-meat contamination event. As an important control, historical S. Montevideos from within this clone were included that retained multiple identical PFGE patterns to the spiced-meat outbreak strains and were isolated from a variety of foods unassociated with this outbreak such as pistachios, chicken, Italian cheese, and several fishes from Indo-China. It is important to note that all of the clinical isolates included here (Figure 3) were collected in association with the spiced-meat outbreak event.


High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach.

Allard MW, Luo Y, Strain E, Li C, Keys CE, Son I, Stones R, Musser SM, Brown EW - BMC Genomics (2012)

Phylogenetic diversity and relationships among a single S. Montevideo clone. GARLI phylogenetic analysis of the outbreak isolates was performed on a set of 43 concatenated ORFs containing informative SNPs (Table 3). Terminal names, scale bar, branch lengths and bootstrap scores are as in Figure 1. Numbers above the branches represent unique SNPs that define these internal branches. The phylogenetic analysis reported here partitions the S. Montevideo clone into 6 lineages (A-F) and expands upon a previous tree [5] with the inclusion of 5 more strains and the noted expansion of outbreak strains into clade E. To the right of the tree, each isolate is labeled with the Not1 pattern that was determined using PFGE with each unique number identifying a new Not1 pattern.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368722&req=5

Figure 3: Phylogenetic diversity and relationships among a single S. Montevideo clone. GARLI phylogenetic analysis of the outbreak isolates was performed on a set of 43 concatenated ORFs containing informative SNPs (Table 3). Terminal names, scale bar, branch lengths and bootstrap scores are as in Figure 1. Numbers above the branches represent unique SNPs that define these internal branches. The phylogenetic analysis reported here partitions the S. Montevideo clone into 6 lineages (A-F) and expands upon a previous tree [5] with the inclusion of 5 more strains and the noted expansion of outbreak strains into clade E. To the right of the tree, each isolate is labeled with the Not1 pattern that was determined using PFGE with each unique number identifying a new Not1 pattern.
Mentions: The importance of NGS in ascertaining high-resolution phylogenetic and molecular epidemiological histories of infectious outbreak clones of bacterial pathogens has recently been noted [18,20]. In the current study, NGS was applied for reconstructing the detailed evolutionary genetic structure of an individual clone of S. Montevideo that is largely indistinguishable using PFGE. Specifically, NGS analysis was applied to a set of S. Montevideo isolates either associated with or genetically homologous to a food contamination event of spiced Italian-style meats in the U.S. in 2009 and 2010 http://www.cdc.gov/Salmonella/montevideo/index.html. We reported previously on the success of NGS for distinguishing some of these isolates from other clonally related isolates unassociated with this spiced-meat S. Montevideo outbreak [5]. Herein, we combined the genomes of 34 highly homogeneous S. Montevideos from food, environmental, and clinical sources from this spiced-meat outbreak with 24 newly sequenced (~15X) S. Montevideo genomes derived from clinical-food matches associated with the same spiced-meat contamination event. As an important control, historical S. Montevideos from within this clone were included that retained multiple identical PFGE patterns to the spiced-meat outbreak strains and were isolated from a variety of foods unassociated with this outbreak such as pistachios, chicken, Italian cheese, and several fishes from Indo-China. It is important to note that all of the clinical isolates included here (Figure 3) were collected in association with the spiced-meat outbreak event.

Bottom Line: In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data.Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications.This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Regulatory Science, Center for Food Safety & Applied Nutrition, U,S, Food & Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740, USA. Marc.Allard@fda.hhs.gov

ABSTRACT

Background: Next-Generation Sequencing (NGS) is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study.

Results: Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data.

Conclusions: Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications. When draft genomes are collected at 15×-20× coverage and passed through a quality filter as part of a data analysis pipeline, including sub-passaged replicates defined by a few SNPs, they can be accurately placed in a phylogenetic context. This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.

Show MeSH
Related in: MedlinePlus