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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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Genome size variation and estimated N50 sizes within Salmonella Montevideo draft genome sequences. The estimated N50 value is a rough estimate of the quality and coverage of the draft genomes which was sequenced to approximately 15-20× coverage. The N50 value represents the average contig size after assembly with the Newbler software. Isolate names correspond to samples in Table 1. Genome length ranged from less than 4.45 mbp to about 4.75 mbp, with most isolates approximately 4.65 mbp in size (unlabeled boxes). Only the larger or smaller genomes are listed.
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Figure 2: Genome size variation and estimated N50 sizes within Salmonella Montevideo draft genome sequences. The estimated N50 value is a rough estimate of the quality and coverage of the draft genomes which was sequenced to approximately 15-20× coverage. The N50 value represents the average contig size after assembly with the Newbler software. Isolate names correspond to samples in Table 1. Genome length ranged from less than 4.45 mbp to about 4.75 mbp, with most isolates approximately 4.65 mbp in size (unlabeled boxes). Only the larger or smaller genomes are listed.

Mentions: Pairwise SNP variation between these four S. Montevideo lineages is listed in Table 2. Intra-serovar SNP diversity was remarkable among the four diverged S. Montevideo genome lineages ranging from 17,600 SNPs (clade I/clade II) to 23,800 SNPs (clade II/clade IV). This latter distance was astonishing given that SNP divergence between S. Montevideo lineage I and S. Pomona, a different group I serovar, falls well within this range (i.e., 22,700 SNPs). In addition to the substantial SNP-based diversity noted among S. Montevideo lineages, genome size also fluctuated widely within this serovar (Figure 2). That is, genome length ranged from less than 4.45 million bp to about 4.75 million bp, sorting largely along intra-serovar clade divisions revealed in the phylogenetic tree (Figure 1). Most of the observed genome size differences appear to be due to the presence or absence of phage elements. The CA clinical isolate 157, for example, is bigger than the outbreak cluster in general due to phage D6. In addition, S. Montevideo strain 163 appears to be enlarged due to insertion of a plasmid pRA1, while strain 206 retains an uncharacterized phage-like sequence and elements of the SPI-7 pathogenicity island. Conversely, two smaller S. Montevideo genomes, 162 and 205, appear to be missing the putative Salmonella phage sequence relative to the outbreak cluster (i.e., clade IV, Figure 1). Akin to findings reported previously on the stress-induced acquisition and loss of phage elements in the Salmonella genome [26], these data signal an important role for insertions and deletions in the diversification of specific clones of S. Montevideo, and, taken together with the above SNP findings, point to a serovar of non-typhoidal Salmonella comprised of several genomically diverged and phylogenetically distinct clones [27-29].


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)

Genome size variation and estimated N50 sizes within Salmonella Montevideo draft genome sequences. The estimated N50 value is a rough estimate of the quality and coverage of the draft genomes which was sequenced to approximately 15-20× coverage. The N50 value represents the average contig size after assembly with the Newbler software. Isolate names correspond to samples in Table 1. Genome length ranged from less than 4.45 mbp to about 4.75 mbp, with most isolates approximately 4.65 mbp in size (unlabeled boxes). Only the larger or smaller genomes are listed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Genome size variation and estimated N50 sizes within Salmonella Montevideo draft genome sequences. The estimated N50 value is a rough estimate of the quality and coverage of the draft genomes which was sequenced to approximately 15-20× coverage. The N50 value represents the average contig size after assembly with the Newbler software. Isolate names correspond to samples in Table 1. Genome length ranged from less than 4.45 mbp to about 4.75 mbp, with most isolates approximately 4.65 mbp in size (unlabeled boxes). Only the larger or smaller genomes are listed.
Mentions: Pairwise SNP variation between these four S. Montevideo lineages is listed in Table 2. Intra-serovar SNP diversity was remarkable among the four diverged S. Montevideo genome lineages ranging from 17,600 SNPs (clade I/clade II) to 23,800 SNPs (clade II/clade IV). This latter distance was astonishing given that SNP divergence between S. Montevideo lineage I and S. Pomona, a different group I serovar, falls well within this range (i.e., 22,700 SNPs). In addition to the substantial SNP-based diversity noted among S. Montevideo lineages, genome size also fluctuated widely within this serovar (Figure 2). That is, genome length ranged from less than 4.45 million bp to about 4.75 million bp, sorting largely along intra-serovar clade divisions revealed in the phylogenetic tree (Figure 1). Most of the observed genome size differences appear to be due to the presence or absence of phage elements. The CA clinical isolate 157, for example, is bigger than the outbreak cluster in general due to phage D6. In addition, S. Montevideo strain 163 appears to be enlarged due to insertion of a plasmid pRA1, while strain 206 retains an uncharacterized phage-like sequence and elements of the SPI-7 pathogenicity island. Conversely, two smaller S. Montevideo genomes, 162 and 205, appear to be missing the putative Salmonella phage sequence relative to the outbreak cluster (i.e., clade IV, Figure 1). Akin to findings reported previously on the stress-induced acquisition and loss of phage elements in the Salmonella genome [26], these data signal an important role for insertions and deletions in the diversification of specific clones of S. Montevideo, and, taken together with the above SNP findings, point to a serovar of non-typhoidal Salmonella comprised of several genomically diverged and phylogenetically distinct clones [27-29].

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