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Single-molecule optical genome mapping of a human HapMap and a colorectal cancer cell line.

Teo AS, Verzotto D, Yao F, Nagarajan N, Hillmer AM - Gigascience (2015)

Bottom Line: Maps were aligned to the human reference by OPTIMA, a new glocal alignment method.Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software.Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies.

View Article: PubMed Central - PubMed

Affiliation: Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, 60 Biopolis Street, Singapore, 138672 Singapore.

ABSTRACT

Background: Next-generation sequencing (NGS) technologies have changed our understanding of the variability of the human genome. However, the identification of genome structural variations based on NGS approaches with read lengths of 35-300 bases remains a challenge. Single-molecule optical mapping technologies allow the analysis of DNA molecules of up to 2 Mb and as such are suitable for the identification of large-scale genome structural variations, and for de novo genome assemblies when combined with short-read NGS data. Here we present optical mapping data for two human genomes: the HapMap cell line GM12878 and the colorectal cancer cell line HCT116.

Findings: High molecular weight DNA was obtained by embedding GM12878 and HCT116 cells, respectively, in agarose plugs, followed by DNA extraction under mild conditions. Genomic DNA was digested with KpnI and 310,000 and 296,000 DNA molecules (≥ 150 kb and 10 restriction fragments), respectively, were analyzed per cell line using the Argus optical mapping system. Maps were aligned to the human reference by OPTIMA, a new glocal alignment method. Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software.

Conclusions: Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies. OPTIMA is an efficient new alignment method; our optical mapping data provide a resource for genome structure analyses of the human HapMap reference cell line GM12878, and the colorectal cancer cell line HCT116.

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Related in: MedlinePlus

Representative optical map of GM12878. DNA molecules were stretched and immobilized onto a glass MapCard surface with the aid of a channel-forming device, cut by KpnI, stained, and visualized by fluorescence imaging. Interrupted linear stretches indicate DNA digested by KpnI. Whirly, non-linear, short, and disjointed DNA molecules are filtered out by the image processing software
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Fig1: Representative optical map of GM12878. DNA molecules were stretched and immobilized onto a glass MapCard surface with the aid of a channel-forming device, cut by KpnI, stained, and visualized by fluorescence imaging. Interrupted linear stretches indicate DNA digested by KpnI. Whirly, non-linear, short, and disjointed DNA molecules are filtered out by the image processing software

Mentions: Argus MapCards were assembled following the manufacturer’s protocol, using Argus consumables and reagents (OpGen Inc). HMW DNA prepared as described above was allowed to flow through a high density channel-forming device (CFD), which was placed on an Argus MapCard surface attached to an Argus MapCard II. This resulted in single DNA molecules being stretched and immobilized on the surface. The CFD was removed, a cap was placed over the DNA, and reagents (antifade, buffer, enzyme, stain) were loaded into the MapCard reservoirs. The assembled MapCard was placed in the Argus MapCard Processor where digestion with KpnI enzyme (Table 1) and staining of DNA molecules occurred in an automated process. The MapCard was removed from the Argus Mapcard Processor and sealed, then placed in the Argus Optical Mapper and set up for automatic data collection as described previously [5]. Argus Mapper was used to image DNA molecules and corresponding restriction fragments by fluorescence microscopy (Fig. 1). The Argus System merged images into channel images and labeled DNA molecules of 150 kb to 2 Mb. Restriction enzyme cut sites were detected as gaps in linear DNA molecules, and the size of each restriction fragment between adjacent cut sites was determined. The Mapper filtered out non-linear distorted fragments and small molecules, identified gaps between fragments, and measured the size of retained high quality fragments. Data from DNA molecules with at least 10 fragments and quality scores of 0.2 were collected from 4 and 6 MapCards for GM12878 and HCT116 cell lines, respectively.Table 1


Single-molecule optical genome mapping of a human HapMap and a colorectal cancer cell line.

Teo AS, Verzotto D, Yao F, Nagarajan N, Hillmer AM - Gigascience (2015)

Representative optical map of GM12878. DNA molecules were stretched and immobilized onto a glass MapCard surface with the aid of a channel-forming device, cut by KpnI, stained, and visualized by fluorescence imaging. Interrupted linear stretches indicate DNA digested by KpnI. Whirly, non-linear, short, and disjointed DNA molecules are filtered out by the image processing software
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696294&req=5

Fig1: Representative optical map of GM12878. DNA molecules were stretched and immobilized onto a glass MapCard surface with the aid of a channel-forming device, cut by KpnI, stained, and visualized by fluorescence imaging. Interrupted linear stretches indicate DNA digested by KpnI. Whirly, non-linear, short, and disjointed DNA molecules are filtered out by the image processing software
Mentions: Argus MapCards were assembled following the manufacturer’s protocol, using Argus consumables and reagents (OpGen Inc). HMW DNA prepared as described above was allowed to flow through a high density channel-forming device (CFD), which was placed on an Argus MapCard surface attached to an Argus MapCard II. This resulted in single DNA molecules being stretched and immobilized on the surface. The CFD was removed, a cap was placed over the DNA, and reagents (antifade, buffer, enzyme, stain) were loaded into the MapCard reservoirs. The assembled MapCard was placed in the Argus MapCard Processor where digestion with KpnI enzyme (Table 1) and staining of DNA molecules occurred in an automated process. The MapCard was removed from the Argus Mapcard Processor and sealed, then placed in the Argus Optical Mapper and set up for automatic data collection as described previously [5]. Argus Mapper was used to image DNA molecules and corresponding restriction fragments by fluorescence microscopy (Fig. 1). The Argus System merged images into channel images and labeled DNA molecules of 150 kb to 2 Mb. Restriction enzyme cut sites were detected as gaps in linear DNA molecules, and the size of each restriction fragment between adjacent cut sites was determined. The Mapper filtered out non-linear distorted fragments and small molecules, identified gaps between fragments, and measured the size of retained high quality fragments. Data from DNA molecules with at least 10 fragments and quality scores of 0.2 were collected from 4 and 6 MapCards for GM12878 and HCT116 cell lines, respectively.Table 1

Bottom Line: Maps were aligned to the human reference by OPTIMA, a new glocal alignment method.Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software.Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies.

View Article: PubMed Central - PubMed

Affiliation: Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, 60 Biopolis Street, Singapore, 138672 Singapore.

ABSTRACT

Background: Next-generation sequencing (NGS) technologies have changed our understanding of the variability of the human genome. However, the identification of genome structural variations based on NGS approaches with read lengths of 35-300 bases remains a challenge. Single-molecule optical mapping technologies allow the analysis of DNA molecules of up to 2 Mb and as such are suitable for the identification of large-scale genome structural variations, and for de novo genome assemblies when combined with short-read NGS data. Here we present optical mapping data for two human genomes: the HapMap cell line GM12878 and the colorectal cancer cell line HCT116.

Findings: High molecular weight DNA was obtained by embedding GM12878 and HCT116 cells, respectively, in agarose plugs, followed by DNA extraction under mild conditions. Genomic DNA was digested with KpnI and 310,000 and 296,000 DNA molecules (≥ 150 kb and 10 restriction fragments), respectively, were analyzed per cell line using the Argus optical mapping system. Maps were aligned to the human reference by OPTIMA, a new glocal alignment method. Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software.

Conclusions: Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies. OPTIMA is an efficient new alignment method; our optical mapping data provide a resource for genome structure analyses of the human HapMap reference cell line GM12878, and the colorectal cancer cell line HCT116.

Show MeSH
Related in: MedlinePlus