Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in Solanum species

• Published in: MethodsX 2018-01, Vol.5, p.328-336
• Main Authors: Gaiero, Paola, Šimková, Hana, Vrána, Jan, Santiñaque, Federico F., López-Carro, Beatriz, Folle, Gustavo A., van de Belt, José, Peters, Sander A., Doležel, Jaroslav, de Jong, Hans
• Format: Article
• Language: English
• Subjects: BioNano genome mapping; Chemistry; Flow sorting; Genome finishing; HMW DNA isolation
• ISSN: 2215-0161; 2215-0161
• DOI: 10.1016/j.mex.2018.03.009

Nuclei sorting and High Molecular Weight (HMW) DNA isolation workflow. Nuclei suspensions are prepared by homogenization or chopping of root tips from germinated seeds or leaves from seedlings or plants, either potted or in vitro. Nuclei are sorted by selecting the G1-S-G2 populations through a FACS Aria or FACS Vantage flow sorter. Flow sorted nuclei are embedded in agarose plugs and HMW DNA is purified with proteinase K and RNAse A and then isolated. Pulsed-field gel electrophoresis (PFGE) is used for DNA quality control. HMW DNA is subsequently label and analyzed on the Irys platform. Optical mapping (OM) data are then processed for de novo OM and hybrid assemblies. [Display omitted] Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the ‘nearly-finished’ level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on •Improving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets.•Obtaining nuclei suspensions through either lab homogenizers or by chopping.•Increasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488?nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. The obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.