Advances in DNA sequencing have made it easier to sequence and assemble plant genomes. Here, we extend an earlier study, and compare recent methods for long read sequencing and assembly. Updated Oxford Nanopore Technology software improved assemblies. Using more accurate sequences produced by repeated sequencing of the same molecule (Pacific Biosciences HiFi) resulted in less fragmented assembly of sequencing reads. Using data for increased genome coverage resulted in longer contigs, but reduced total assembly length and improved genome completeness. The original model species, Macadamia jansenii, was also compared with three other Macadamia species, as well as avocado (Persea americana) and jojoba (Simmondsia chinensis). In these angiosperms, increasing sequence data volumes caused a linear increase in contig size, decreased assembly length and further improved already high completeness. Differences in genome size and sequence complexity influenced the success of assembly. Advances in long read sequencing technology continue to improve plant genome sequencing and assembly. However, results were improved by greater genome coverage, with the amount needed to achieve a particular level of assembly being species dependent.

High-quality DNA and RNA forms the basis of genomic and genetic investigations. The extraction of DNA and RNA from woody trees, like avocado (Persea americana Mill.), is challenging due to compounds which interact with nucleic acids and influence separation. Previously reported methods of DNA and RNA extraction from avocado have issues of low yield, quality and applicability across different cultivars and tissue types. In the current study, methods have been optimised for high-quality DNA extraction from 40 avocado cultivars and RNA extraction from multiple tissue types, including roots, stem, leaves, flowers and fruits. The method is based on the modification of the cetyltrimethylammonium bromide buffer, centred around the specific optimisation of chemicals, such as sodium dodecyl sulphate, polyvinylpyrrolidone, sodium sulphite, polyethylene glycol and β-mercaptoethanol. The DNA extraction method yielded high-molecular weight DNA from the leaf tissue of 40 avocado cultivars belonging to Mexican, Guatemalan and West Indian avocado horticultural groups. The method was further optimised for RNA extraction from different avocado plant parts, enabling extraction using amounts as low as ~10 mg of starting material. The DNA and RNA extracted was successfully used for long- and short-read sequencing and gene expression analysis. The methods developed may also be applicable to other recalcitrant plant species.