Labelled and stained DNA was loaded on the Irys chip, and four consecutive runs were performed (each run consisting of 30 cycles). A total of 489.8 Gb data were generated, of which 221.7 Gb exceeded 150 kb, the threshold for map assembly. These filtered data (>150 kb), corresponding to 401× coverage of the subterranean clover genome, were compiled from 994 895 molecules with N50 of 215.4 kb. De novo assembly of the BioNano map was performed by a pairwise comparison of all single molecules and graph building (Cao et al., 2014). A P‐value threshold of 1e−9 was used during the pairwise assembly, 1e−10 for extension and refinement steps and 1e−15 for a final refinement.