Irys platform

High-molecular weight (HMW) DNA was extracted from up to 5 g fresh leaf tissue using a modified Bionano Genomics protocol^{2 (link)}. Briefly, extracted HMW DNA was nicked with the enzyme Nt.BspQI (NEB, Beverly, MA, USA), fluorescently labeled, repaired, and stained overnight according to the Bionano Genomics nick-labeling protocol^{31 (link)}. KBS-Mac-74 nick-labeled DNA was run on a single flow cell on the Irys platform (Bionano Genomics, San Diego, CA, USA), for 90 cycles to generate 22.5 Gb raw data. The IrysView software (Bionano Genomics; version 2.5.1) was used to quality filter the raw data (>100 kb length, >2.75 signal/noise ratio) and molecules were assembled into contigs using the default “human genome” parameters. Resulting Bionano cmaps were compared against the different assemblies using Bionano RefAlign^{2 (link)}, and collapsed regions or artificial expansions were detected as structural variations using the structomeIndel.py script (https://github.com/RyanONeil/structome). Rates for FP and FN nicking sites were extracted from the .err files, and genome coverage was computed from the .xmap files.

Strains Z1723 and Z1767 were cultured from a single colony in LB medium to an OD₆₀₀=0.7. 1 ml of cells/agarose plug were harvested (4000 g, 5 min) and intact chromosomes were extracted according to the Bionano Prep Cell Culture DNA Isolation Protocol (Bionano). Briefly, harvested cells were washed twice in Bionano Cell Buffer (Bionano). Washed cells were embedded in 2% low-melt agarose plugs and cells were lysed (1 h at 37 °C) with lysozyme enzyme (100 µl) using CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad). DNA containing plugs were washed twice with nuclease free water then treated with Proteinase K (Qiagen) in Bionano Lysis Buffer according to the Bionano Prep Cell Culture DNA Isolation Protocol. All subsequent procedure steps (RNase treatment, DNA extraction, quantitation and labelling) and optical mapping on Bionano Irys platform were provided as a service by Earlham Institute (Norwich, UK). Structural variant analysis was provided by Bionano and structural variant maps visualized using Bionano access (Bionano).