Vitis vinefera L. DNA preparation for third-generation sequencing

Abstract Several approaches have been proposed to sequence the genome of Vitis vinifera L. (grapevine), utilizing low-resolution second-generation platforms that lead to partial coverage and low-quality reads. Nevertheless, the characterization of the grapevine genetic resources could be better addressed through extensive and high-resolution genome sequencing. MinION is a third-generation sequencer preferred by many laboratories due to its relatively low cost, ease of use, and small size. To exploit MinION at its full potential, it requires high-quality DNA which is difficult to obtain in grapevine. Here we establish a workflow for DNA for MinION sequencing long reads from grapevine. This protocol was tested with undeveloped leaves, apex, helices, petioles, and 4th leaves samples. In addition, a circulomic size selection approach was tested to provide an unparalleled size of DNA reads. Our results suggest that longer reads of grapevine DNA associate with the use of fresh undeveloped tissue and adjusted conditions for the purification of cell nuclei. Short read elimination protocol increased N50 read length with a reasonable compromise for overall data yield. Our workflow presents a significant advancement for long-read quality DNA isolation for grapevine, providing also technical considerations for high-quality DNA preparation compatible with MinION from plants.