A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants

Abstract

Background: PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing.

Results: We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively.

Conclusions: DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

Keywords: DNA extraction; Genome sequencing; Gesneriaceae; Long-read sequencing; Next-generation sequencing; PacBio HiFi; SMRTbell™ library; Streptocarpus.

Fig. 1

Quality of small scale DNA extractions using the protocol developed in this study. DNA was extracted in triplicates from two species of Streptocarpus, S. grandis and S. kentaniensis, using two different lysis conditions: 4H—4 h at 58 °C, ON—overnight at 50 °C. a Scatter plot of TapeStation Genomic DIN values (y-axis) versus obtained DNA in ng per g leaf sample (x-axis). b Scatter plot of spectrophotometer ratios, A260/A230 (A260vsA230) versus A260/A280 (A260vsA280). c Scatter plot of DNA concentration (ng/µl) quantified with fluorometer (Qubit_conc) versus spectrophotometer (Nanodrop_conc). d Scatter plot of Femto Pulse GQN analyses. GQN values (y-axis) indicate the proportion of the fragment quantity of the total DNA quantity of the GQNxkb threshold length (x-axis). For example, 50 on the x-axis indicates a fragment size of GQN_50kb and the proportion of DNA quantity > 50 kb is shown on the y-axis. e TapeStation gel image of extracted DNA. Lanes: A1 ladder, B1-G1 S. grandis, H1-E2 S. kentaniensis, B1-D1 and H1-B2 ON lysis, E1-G1 and C2-E2 4H lysis

Fig. 2

Representative results of Femto Pulse runs on Streptocarpus small scale DNA extracted in this study. a, b S. grandis c, d S. kentaniensis a, c Lysis condition overnight at 50 °C b, d Lysis condition for 4 h at 58 °C

Fig. 3

Schematic overview illustration of the DNA extraction method for PacBio HiFi sequencing developed in this study

Fig. 4

Schematic illustration of the steps involved in the PacBio HiFi DNA extraction protocol. Steps a–o explained in text under ‘Procedure’