You are here : Home > Research Centers and Units > Genoscope > Laboratory of Molecular Biology Methods for Genomics

Laboratory

Laboratory of Molecular Biology Methods for Genomics

Activities


Published on 8 October 2019

Preparation of very high molecular weight DNA 

Purifying very high molecular weight DNA (> 200 kb) is currently a real need in order to meet the requirements of new technologies such as optical mapping, as is the construction of databanks for long- read sequencing approaches.

Our objective is to acquire know-how that will enable us to propose DNA preparation solutions appropriate to each type of organism and the quality and quantity required for implementation of the studies conducted by the Genoscope platform in the context of collaborative projects.

LBioMEG has an OpGen Argus® system (Figure 1A) (www.opgen.com) and a BioNano Genomics Irys™ system (figure 2A) (www.bionanogenomics.com) enabling semi-automated implementation of genomic optical maps. The OpGen Argus® system is used for genomes of less than 20 MB while the BioNano Genomics Irys™ system is used for genomes greater than 20 MB.​

.

 

Figure 1 A. The « Argus™ Optical Mapping System ». B. Generation of a whole-genome restriction map is implemented by digestion of linear DNA fragments immobilized on glass slides.​






The principle of the optical map data is based on establishing an enzymatic profile of genomic DNA molecules (gDNA) based on the size and order of the fragments obtained by digestion. The compilation of several profiles derived from several molecules enables compilation of a complete restriction map of a genome known as a whole-genome map (Figure 3).

The two systems use different approaches to generate the optical maps. With the OpGen Argus® system, the gDNA fragments are rendered linear and then immobilized on a glass slide before being digested by a restriction enzyme (Figure 1B). With the BioNano Genomics Irys™ system, the gDNA fragments are labeled using fluorescent Taq polymerase and dNTP after digestion of the gDNA by a restriction endonuclease (Figure 2B). The labeled gDNA fragments are then known as NLRS (« Nicking, Labelling, Repair and Staining ») DNA. Subsequently, the NLRS gDNA are loaded and linearized over several cycles in nanochannels (Figure 2B).


Figure 2 A . The « Irys™ BioNano Genomics system. » B. A restriction map is generated by loading and linearizing the labeled gDNA (NLRS gDNA) in nanochannels over several cycles.

 

 

 

 

 

 





Figure 3 . The optical map of the genome is implemented by assembling the molecules.

The difficulty of obtaining a whole-genome optical map resides in the preparation of the starting material. It is essential to use material (gDNA) of very high quality. The gDNA molecules obtained after extraction must be of very high molecular weight (~ 200-400 kb). For organisms with a genome greater than 7 Mb, it is recommended to extract the gDNA from lysed cells trapped in agar (agar plugs).

The applications of an optical map are multiple. With regard to bacterial genomes, the most frequent uses are typing (Figure 4A) and comparative genomics (Figure 4B) of strains. But optical maps also enable validation and improvement of sequence assembly (Figure 4C). With the BioNano Genomics Irys™ system, the optical maps also enable detection of structural variants and detection of copy-number variations (CNV) (Figure 4D and 4E).

In the context of its collaborations, LBIOMEG makes its skills available to the scientific community for the implementation and analysis of optical maps of genomes of interest.


 Figure 4. ​​The ordered restriction map of a complete genome enables typing of strains (A), comparative genomics (B), improvement of assemblies (C), detection of structural variants (D) and detection of copy-number variations (CNV) (D).