The genome sequence of subterranean clover (TSUd_r1.1) (Hirakawa et al., 2016) was analysed with Nickers software to assess the frequency of recognition sites for four different nicking enzymes (Nt.BbvC1, Nt.BsPQ1, Nb.bsm1 and Nb.BsrD1). The optimal labelling frequency was calculated for endonuclease Nt.BsPQ1 (7.6 sites/100 kb). DNA was then processed using NLRS protocol using the IrysPrep® Reagent Kit (BioNano Genomics, San Diego, CA) following manufacturer's instructions. DNA was nicked using 8U of Nt.BspQ1 (New England BioLabs, Beverly, MA) for two hours at 37 °C in NEBuffer 3. The nicked DNA was labelled with a fluorescent‐dUTP nucleotide analogue using Taq polymerase (New England BioLabs) for one hour at 72 °C. After labelling, the nicks were ligated with Taq ligase (New England BioLabs) in the presence of dNTPs for 30 min at 37 °C. The backbone of the labelled DNA was stained with IrysPrep® DNA Stain (BioNano Genomics). The NLRS DNA concentration was measured again with the Quant‐iTTM PicoGreen® dsDNA assay.