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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 of Salmonella Montevideo based on a GARLI analysis of 72,063 variable SNP sites of which 63,987 were identified as parsimony informative. The tree was rooted with four outgroups including S. Schwarzengrund, S. Pomona, and S. Javiana. Terminal names correspond to samples in Table 1. The numbers at the base of each node are bootstrap scores with most of the deepest nodes supported at 100%. The scale bar units are nucleotide substitutions per site and these are proportional across the branch lengths with longer branches having greater substitutions. S. Montevideo strains partitioned into four clades designated I-IV.
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Figure 1: Phylogenetic diversity of Salmonella Montevideo based on a GARLI analysis of 72,063 variable SNP sites of which 63,987 were identified as parsimony informative. The tree was rooted with four outgroups including S. Schwarzengrund, S. Pomona, and S. Javiana. Terminal names correspond to samples in Table 1. The numbers at the base of each node are bootstrap scores with most of the deepest nodes supported at 100%. The scale bar units are nucleotide substitutions per site and these are proportional across the branch lengths with longer branches having greater substitutions. S. Montevideo strains partitioned into four clades designated I-IV.

Mentions: In order to explore the evolutionary genetic diversity of Salmonella Montevideo, NGS analysis was performed on 47 strains of this serovar (Table 1). This included assembling the raw reads to form contigs of overlapping sequence, annotating those contigs to determine which genes were present, and then determining homology among genes and aligning and concatenating those genetic elements for population and phylogenetic analyses. Roche-Titanium whole-genome shotgun sequencing technology [23,24] provided 15-20× coverage for each genome reported, and downstream contig assembly and sequence alignment provided over 4.5-5 mbp of assembled contigs for each isolate. Additional data filtering yielded 72,063 variable SNP sites of which 63,987 were identified as parsimony informative (i.e., SNPs shared by two or more strains in the alignment) and subjected to phylogenetic analysis on the FDA bioinformatics, Linux based computer cluster using likelihood and parsimony methods. The resultant evolutionary tree derived from the informative SNP data yielded two important observations (Figure 1). First, S. Montevideo formed a monophyletic group of strains phylogenetically distinct from other neighboring serovars including S. Schwarzengrund, S. Pomona, and S. Javiana. Second, S. Montevideo strains partitioned into four disparate clades (designated I-IV), several of which were defined by a mixture of both natural and laboratory isolates. Clade III, for example, comprised a clinical isolate associated with tomato (206_Clinical) as well as a single strain (160_Clinical_FL) from the widely characterized subspecies I Salmonella Reference collection, SARB [25].


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 of Salmonella Montevideo based on a GARLI analysis of 72,063 variable SNP sites of which 63,987 were identified as parsimony informative. The tree was rooted with four outgroups including S. Schwarzengrund, S. Pomona, and S. Javiana. Terminal names correspond to samples in Table 1. The numbers at the base of each node are bootstrap scores with most of the deepest nodes supported at 100%. The scale bar units are nucleotide substitutions per site and these are proportional across the branch lengths with longer branches having greater substitutions. S. Montevideo strains partitioned into four clades designated I-IV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Phylogenetic diversity of Salmonella Montevideo based on a GARLI analysis of 72,063 variable SNP sites of which 63,987 were identified as parsimony informative. The tree was rooted with four outgroups including S. Schwarzengrund, S. Pomona, and S. Javiana. Terminal names correspond to samples in Table 1. The numbers at the base of each node are bootstrap scores with most of the deepest nodes supported at 100%. The scale bar units are nucleotide substitutions per site and these are proportional across the branch lengths with longer branches having greater substitutions. S. Montevideo strains partitioned into four clades designated I-IV.
Mentions: In order to explore the evolutionary genetic diversity of Salmonella Montevideo, NGS analysis was performed on 47 strains of this serovar (Table 1). This included assembling the raw reads to form contigs of overlapping sequence, annotating those contigs to determine which genes were present, and then determining homology among genes and aligning and concatenating those genetic elements for population and phylogenetic analyses. Roche-Titanium whole-genome shotgun sequencing technology [23,24] provided 15-20× coverage for each genome reported, and downstream contig assembly and sequence alignment provided over 4.5-5 mbp of assembled contigs for each isolate. Additional data filtering yielded 72,063 variable SNP sites of which 63,987 were identified as parsimony informative (i.e., SNPs shared by two or more strains in the alignment) and subjected to phylogenetic analysis on the FDA bioinformatics, Linux based computer cluster using likelihood and parsimony methods. The resultant evolutionary tree derived from the informative SNP data yielded two important observations (Figure 1). First, S. Montevideo formed a monophyletic group of strains phylogenetically distinct from other neighboring serovars including S. Schwarzengrund, S. Pomona, and S. Javiana. Second, S. Montevideo strains partitioned into four disparate clades (designated I-IV), several of which were defined by a mixture of both natural and laboratory isolates. Clade III, for example, comprised a clinical isolate associated with tomato (206_Clinical) as well as a single strain (160_Clinical_FL) from the widely characterized subspecies I Salmonella Reference collection, SARB [25].

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