Long read sequencing

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.

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The standard protocol of Oxford Nanopore Technologies (ONT) ligation sequencing kit (e.g. SQK-LSK109) uses AMPure beads at DNA repair and end-prep clean up and adapter clean up steps. AMPure beads either shear long fragments DNA or form clump with ultra-High Molecular Weight DNA and High Molecular Weight DNA resulting poor sequencing library preparation. In this method, AMPure beads are replaced by the PEG/NaCl solution and short read eliminator (SRE) XL kit to keep DNA intact during the sequencing library preparation. In addition, loading beads (LB) is also excluded on preparing loading library in order to prevent beads clumping. This protocol is useful to generate long 426 kb ultra-long reads with 7.2 Gb sequence throughput (in MinION) generated in 24 h.

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We report a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION) and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of PacBio and Nanopore reads. The assemblies were compared for contiguity, accuracy and completeness as well as sequencing costs and DNA material requirements. Overall, the three long read technologies produced highly contiguous and complete genome assemblies of Macadamia jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs. 

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