In brief

Long read sequencing technologies require highly pure high molecular weight (HMW) intact DNA to utilise the optimal capacity of these technologies. Getting pure HMW DNA in non-model plant is a challenge due to complex cellular metabolites that chemically behave like DNA molecules. Isolation of nuclei helps to remove the majority of cellular impurities in DNA extraction processes. Recently, few protocols for HMW DNA extraction from plant species targeting to long read sequencing have been developed from different laboratories. Herein, the HMW nuclear DNA extraction method is optimised at the nuclei isolation step where the centrifugation speed is independent of genome size. Using this method, highly pure HMW nuclear DNA was extracted from two difficult plant species with a wide range of genome size – Macadamia jansenii (~800 mb) and Beauregard sweet potato (~2.4 Gb) and the quality was assessed by nanopore sequencing.


  • Using this method, highly pure nuclear HMW DNA suitable for a long read sequencing can be extracted in 5-6 h from difficult plant samples.

  • This described method does not require prior knowledge of genome size and optimisation of centrifugation speed during nuclei isolation therefore users can save optimisation time and materials while working with a new plant species.
  • High throughput data (28 – 93 Gb in PromethION, 5-20 Gb in MinION) with long reads up to 295 kb has been generated in ONT using DNA prepared from this method.