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.
ABSTRACT
Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929-Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii. This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.