Intact Genomic DNA Purified from Flow-sorted Nuclei Unleashes the Potential of Nanochannel-based Optical Mapping in Solanum Species

Optical mapping of high molecular weight DNA (HMW DNA) through nanochannels is the ideal tool for completing genome assemblies by sizing and/or closing gaps, ordering contigs and scaffolds, joining scaffolds, correcting assembly errors, evaluating and anchoring the assembly and even identifying, spanning and assembling repeated sequences. So far this strategy has been applied in sequencing efforts of bacteria and other low complexity genomes, while major developments have been achieved for the human genome sequence and some large complex genomes (e.g. domesticated goat). In the higher plant genomics community, researchers still face the challenges of obtaining sufficient amounts of purified HMW DNA due to the thick cell walls and cytoplasmic polyphenols and polysaccharides. Here we describe a flow sorting method for isolation of highly purified interphase nuclei from small amounts of fast growing root or leaf material, using different flow sorting platforms and DNA stains. The nuclei thus obtained are embedded in agarose plugs and treated by proteinase to obtain very pure HMW DNA, suitable for nanochannel-based optical mapping. The method has been applied to tomato Heinz 1706, obtaining high quality DNA that allowed for a successful nanochannel-based optical mapping run with good labelling density (7.7 sites per 100 Kbp), average molecule length of 290 kb and a throughput of 1.3 Gb per scan. The same method is under way for other Solanum species belonging to the potato clade.