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Whole genome mapping as a fast-track tool to assess genomic stability of sequenced Staphylococcus aureus strains.

Sabirova JS, Xavier BB, Ieven M, Goossens H, Malhotra-Kumar S - BMC Res Notes (2014)

Bottom Line: Of the twelve strains tested, one (USA300_FPR3757) showed a 19-kbp deletion on WGM compared to its in silico generated map and reference sequence data.Frequent subculturing and inter-laboratory transfers can induce genomic and therefore, phenotypic changes that could compromise the utility of standard reference strains.WGM can thus be used as a rapid genome screening method to identify genomic rearrangements whose size and type can be confirmed by sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Vaccine & Infectious Disease Institute, Universiteit Antwerpen, Antwerp, Belgium. surbhi.malhotra@uantwerpen.be.

ABSTRACT

Background: Whole genome (optical) mapping (WGM), a state-of-the-art mapping technology based on the generation of high resolution restriction maps, has so far been used for typing clinical outbreak strains and for mapping de novo sequence contigs in genome sequencing projects. We employed WGM to assess the genomic stability of previously sequenced Staphylococcus aureus strains that are commonly used in laboratories as reference standards.

Results: S. aureus strains (n = 12) were mapped on the Argus™ Optical Mapping System (Opgen Inc, Gaithersburg, USA). Assembly of NcoI-restricted DNA molecules, visualization, and editing of whole genome maps was performed employing MapManager and MapSolver softwares (Opgen Inc). In silico whole genome NcoI-restricted maps were also generated from available sequence data, and compared to the laboratory-generated maps. Strains showing differences between the two maps were resequenced using Nextera XT DNA Sample Preparation Kit and Miseq Reagent Kit V2 (MiSeq, Illumina) and de novo assembled into sequence contigs using the Velvet assembly tool. Sequence data were correlated with corresponding whole genome maps to perform contig mapping and genome assembly using MapSolver. Of the twelve strains tested, one (USA300_FPR3757) showed a 19-kbp deletion on WGM compared to its in silico generated map and reference sequence data. Resequencing of the USA300_FPR3757 identified the deleted fragment to be a 13 kbp-long integrative conjugative element ICE6013.

Conclusions: Frequent subculturing and inter-laboratory transfers can induce genomic and therefore, phenotypic changes that could compromise the utility of standard reference strains. WGM can thus be used as a rapid genome screening method to identify genomic rearrangements whose size and type can be confirmed by sequencing.

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Aligned experimental andin silicowhole genome maps of FPR3757 (A); alignment of experimental map of FPR3757 to sequence contigs (B); alignment ofin silicomap of FPR3757 to sequence contigs (C). Regions highlighted in green indicate perfect alignment. The region corresponding to the deletion is colored in grey and hashed with dark grey diagonal bars in the in silico map.
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Fig2: Aligned experimental andin silicowhole genome maps of FPR3757 (A); alignment of experimental map of FPR3757 to sequence contigs (B); alignment ofin silicomap of FPR3757 to sequence contigs (C). Regions highlighted in green indicate perfect alignment. The region corresponding to the deletion is colored in grey and hashed with dark grey diagonal bars in the in silico map.

Mentions: We utilized WGM to assess the genomic stability of previously sequenced S. aureus that are also commonly used as laboratory reference strains (Additional file 1: Table S1). Comparison of experimental whole genome maps to in silico restriction maps (NcoI) generated from published sequences showed that for majority of the strains (COL, MRSA476, 71193, ED133, JKD6008, JKD6159, LGA251, MRSA252, N315, TCH1516, and HO 5096 0412), both maps were identical (100–99.9% map similarity; Figure 1). The 0.1% difference in in silico and experimental maps of some strains (COL, MRSA476, 71193, ED133, JKD6008, LGA251, MRSA252, N315, and TCH1516) could be attributed to the loss of small DNA fragments in the experimental maps. The rate of loss of fragments ≤ 2 kb could be as high as 75% during mapcard processing comprising in situ restriction, staining and washing of linearized and immobilized DNA molecules, as described previously [9]. However, for S. aureus FPR3757, a clear 1.5% map difference was observed between the experimental and corresponding in silico WGMs. The experimental WGM of FPR3757 displayed a 19-kbp genomic deletion compared to its in silico map (Figure 2). According to published sequence data, the deleted fragment contained genes SAUSA300_1456 to SAUSA300_1474 that included a maltose degradation operon (Figure 3A).Figure 1


Whole genome mapping as a fast-track tool to assess genomic stability of sequenced Staphylococcus aureus strains.

Sabirova JS, Xavier BB, Ieven M, Goossens H, Malhotra-Kumar S - BMC Res Notes (2014)

Aligned experimental andin silicowhole genome maps of FPR3757 (A); alignment of experimental map of FPR3757 to sequence contigs (B); alignment ofin silicomap of FPR3757 to sequence contigs (C). Regions highlighted in green indicate perfect alignment. The region corresponding to the deletion is colored in grey and hashed with dark grey diagonal bars in the in silico map.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197248&req=5

Fig2: Aligned experimental andin silicowhole genome maps of FPR3757 (A); alignment of experimental map of FPR3757 to sequence contigs (B); alignment ofin silicomap of FPR3757 to sequence contigs (C). Regions highlighted in green indicate perfect alignment. The region corresponding to the deletion is colored in grey and hashed with dark grey diagonal bars in the in silico map.
Mentions: We utilized WGM to assess the genomic stability of previously sequenced S. aureus that are also commonly used as laboratory reference strains (Additional file 1: Table S1). Comparison of experimental whole genome maps to in silico restriction maps (NcoI) generated from published sequences showed that for majority of the strains (COL, MRSA476, 71193, ED133, JKD6008, JKD6159, LGA251, MRSA252, N315, TCH1516, and HO 5096 0412), both maps were identical (100–99.9% map similarity; Figure 1). The 0.1% difference in in silico and experimental maps of some strains (COL, MRSA476, 71193, ED133, JKD6008, LGA251, MRSA252, N315, and TCH1516) could be attributed to the loss of small DNA fragments in the experimental maps. The rate of loss of fragments ≤ 2 kb could be as high as 75% during mapcard processing comprising in situ restriction, staining and washing of linearized and immobilized DNA molecules, as described previously [9]. However, for S. aureus FPR3757, a clear 1.5% map difference was observed between the experimental and corresponding in silico WGMs. The experimental WGM of FPR3757 displayed a 19-kbp genomic deletion compared to its in silico map (Figure 2). According to published sequence data, the deleted fragment contained genes SAUSA300_1456 to SAUSA300_1474 that included a maltose degradation operon (Figure 3A).Figure 1

Bottom Line: Of the twelve strains tested, one (USA300_FPR3757) showed a 19-kbp deletion on WGM compared to its in silico generated map and reference sequence data.Frequent subculturing and inter-laboratory transfers can induce genomic and therefore, phenotypic changes that could compromise the utility of standard reference strains.WGM can thus be used as a rapid genome screening method to identify genomic rearrangements whose size and type can be confirmed by sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Vaccine & Infectious Disease Institute, Universiteit Antwerpen, Antwerp, Belgium. surbhi.malhotra@uantwerpen.be.

ABSTRACT

Background: Whole genome (optical) mapping (WGM), a state-of-the-art mapping technology based on the generation of high resolution restriction maps, has so far been used for typing clinical outbreak strains and for mapping de novo sequence contigs in genome sequencing projects. We employed WGM to assess the genomic stability of previously sequenced Staphylococcus aureus strains that are commonly used in laboratories as reference standards.

Results: S. aureus strains (n = 12) were mapped on the Argus™ Optical Mapping System (Opgen Inc, Gaithersburg, USA). Assembly of NcoI-restricted DNA molecules, visualization, and editing of whole genome maps was performed employing MapManager and MapSolver softwares (Opgen Inc). In silico whole genome NcoI-restricted maps were also generated from available sequence data, and compared to the laboratory-generated maps. Strains showing differences between the two maps were resequenced using Nextera XT DNA Sample Preparation Kit and Miseq Reagent Kit V2 (MiSeq, Illumina) and de novo assembled into sequence contigs using the Velvet assembly tool. Sequence data were correlated with corresponding whole genome maps to perform contig mapping and genome assembly using MapSolver. Of the twelve strains tested, one (USA300_FPR3757) showed a 19-kbp deletion on WGM compared to its in silico generated map and reference sequence data. Resequencing of the USA300_FPR3757 identified the deleted fragment to be a 13 kbp-long integrative conjugative element ICE6013.

Conclusions: Frequent subculturing and inter-laboratory transfers can induce genomic and therefore, phenotypic changes that could compromise the utility of standard reference strains. WGM can thus be used as a rapid genome screening method to identify genomic rearrangements whose size and type can be confirmed by sequencing.

Show MeSH
Related in: MedlinePlus