Modified CTAB protocols for high‐molecular‐weight DNA extractions from ferns

Abstract

Premise Efficient protocols for extracting high‐molecular‐weight (HMW) DNA from ferns facilitate the long‐read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)‐based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time. Methods and Results We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A260/A230 and A260/A280 > 1.8). Conclusions This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.

Organellar phylogenomics of Ophioglossaceae fern genera

“…The most promising way to sequence a fern mitogenome is to adapt the long-read NGS approaches. For this, both an efficient protocol of high-molecular-weight DNA extraction (e.g., Xie et al., 2023 ) and a long-read sequencing strategy (e.g., Nanopore) are indispensable. Therefore, we are looking forward to future attempts of long-read sequencing and assembly of fern mitogenomes, which will facilitate publication of mitogenome sequences across fern diversity.…”

Emerging methods in botanical DNA/RNA extraction

“…Many fern lineages have notoriously large genomes, which necessitates modifications to DNA extraction protocols to ensure sufficient quantities of high‐molecular‐weight DNA to complete long‐read sequencing. Xie et al (2023) presented two modifications to standard CTAB DNA extraction protocols that increase DNA yield: a strategy to prevent DNA shearing and a nuclei isolation approach that results in substantially larger quantities of extracted DNA. Although it is established that preventing DNA shearing improves long‐read sequencing performance (Gong et al, 2019), Xie et al (2023) introduce a nuclei isolation step that, while requiring more input tissue, generates an order of magnitude more DNA per extraction than standard approaches.…”

“…Xie et al (2023) presented two modifications to standard CTAB DNA extraction protocols that increase DNA yield: a strategy to prevent DNA shearing and a nuclei isolation approach that results in substantially larger quantities of extracted DNA. Although it is established that preventing DNA shearing improves long‐read sequencing performance (Gong et al, 2019), Xie et al (2023) introduce a nuclei isolation step that, while requiring more input tissue, generates an order of magnitude more DNA per extraction than standard approaches. Two genomes have been sequenced using the protocol modifications developed by Xie et al (2023), and both have read length N50 scores >14 kbp (Wickell et al, 2021; Rahmatpour et al, 2023).…”

“…Although it is established that preventing DNA shearing improves long‐read sequencing performance (Gong et al, 2019), Xie et al (2023) introduce a nuclei isolation step that, while requiring more input tissue, generates an order of magnitude more DNA per extraction than standard approaches. Two genomes have been sequenced using the protocol modifications developed by Xie et al (2023), and both have read length N50 scores >14 kbp (Wickell et al, 2021; Rahmatpour et al, 2023).…”

Balancing read length and sequencing depth: Optimizing Nanopore long‐read sequencing for monocots with an emphasis on the Liliales

“…Previous studies have compared different DNA extraction methods to obtain sequencing data for a variety of plant lineages (e.g., Abdel‐Latif and Osman, 2017; Pipan et al, 2018; Scobeyeva et al, 2018), although many of these were not intended to generate extractions optimized for long‐read sequencing technology, which requires extremely pure, high‐quality DNA. In the past several years, new protocols have been developed for generating high‐molecular‐weight DNA specifically for long‐read sequencing (e.g., Mayjonade et al, 2016; Li et al, 2020; Jones et al, 2021; Maghini et al, 2021; Russo et al, 2021; Kang et al, 2023; Xie et al, 2023); however, most protocols do not test how each modification may impact sequencing results, and not all methods work on plant lineages with complicating factors such as secondary metabolites, succulence, high fiber content, or other structural considerations. A recent study by Russo et al (2022) compared the results of two extraction protocols for long‐read sequencing across multiple lineages of plants, yet the impacts of some modifications were still not explicitly shown.…”