Obtaining high quality DNA from plant tissues for nanopore sequencing

In the final talk of the session Stella Loke from the Deakin Genomics centre in Australia began by discussing the challenges of preparing plant DNA for Nanopore sequencing. Cell walls, waxy cuticles, pigments, polysaccharides, carbohydrates, polyphenolics, proteins and lipids present in plant cells can potentially all cause downstream problems. Stella went on to stress the need for multiple methods to validate the QC metrics: Nanodrop for DNA purity and concentration; Qubit for concentration; tapestation and agarose gels to asses the size of the DNA. If Qubit and Nanodrop closely agree on the concentration this is also a good proxy for a clean DNA sample.

Stella has an optimised pipeline she uses for preparing DNA for nanopore sequencing: Optimised (to each sample) extraction method, DNA clean up, size selection and finally library preparation. Moving onto discuss plant tissue processing, Stella stressed that getting good HMW DNA out of a tissue requires picking a good tissue. For best DNA extraction pick fresh young leaves rather than stems. Leaves can be freeze dried shortly after harvesting or preserved in 80-95% ethanol (with antioxidant) for a short time if required. For tissue homogenisation you can use liquid nitrogen freezing and a pestle and mortar or use large stainless steel ball bearings for bead beating on frozen tissue samples. Stella recommends trying DNA extraction by SDS or CTAB methods but has also had some success with commercial kits. Her CTAB method follows the traditional method with a few tweaks: higher CTAB reagent, higher salt, PVP 10K and 40K. Importantly she uses β-mercaptoethanol to prevent phenolic compounds oxidising and binding to the DNA.

If impurities remain after extraction the DNA can be further cleaned using salt precipitation steps (proteins, carboyhdrates, SDS), extra SPRI bead clean-ups (salt contamination). Some commercial kits also work well to clean DNA of impurities. Stella gave some examples of common contaminants and how to spot them – e.g if the DNA pellet is brown it suggests there are phenolics in sample. Once pure DNA is isolated DNA it can be size selected with a method of choice to enrich for long fragments. Stella uses the Zymo Gel recovery kit as it is focused on HMW DNA isolation. Stella finds that size selection also helps cleaning up the DNA further, getting the sample closer to the ideal nanodrop ratios.

Stella wrapped up her talk by showing some of her highest throughput MinION runs on plant DNA (17.7 and 23.7Gb) and stressing that it is worth the effort and time to put into DNA extraction. Summarising: if you have bad DNA you get bad library prep and bad sequencing as a result.