SILEX: A fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

Abstract

Background The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

Hypermethylation and small RNA expression are associated with increased age in almond (Prunus dulcis[Mill.] D.A. Webb) accessions

“…High-quality DNA was extracted from leaves following a modified version of the protocol outlined in Vilanova et al ., 2020. Briefly, samples were ground to a fine powder with a mortar and pestle in liquid nitrogen, and 50 mg of the ground material was added to 1 mL of extraction buffer (2% w/v CTAB; 2% w/v PVP-40; 20 mM EDTA; 100 mM Tris-HCl [pH 8.0]; 1.4 M NaCl), 14 μL beta-mercaptoethanol, and 2 μL RNase (10 mg/mL).…”

Sticky problems: extraction of nucleic acids from molluscs

“…A modified agarose plug-based approach was used effectively to prepare HMW genomic DNA for long-read sequencing toward characterization of the Pecten maximus (bivalve) genome by, in 2020 [103]. With the increasing popularity of long-read sequencing, alternative methods have been developed to minimize mechanical shearing during extraction in order to prepare HMW DNA, potentially also from molluscs [104,105], and commercial biotech companies offer an ever-growing variety of kits for extraction of HMW DNA [106,107] .…”

A gap-free tomato genome built from complementary PacBio and Nanopore long DNA sequences reveals extensive linkage drag during breeding

“…Whilst this was also rapidly adopted in the field of plant genomics (Vanburen et al, 2015;Zapata et al, 2016), the application of nanopore sequencing developed by Oxford Nanopore Technologies (ONT) led to long read sequencing with minimal capital outlay (Schmidt et al, 2017;Michael et al, 2018). Increases in sequencing yield and improvements in raw base accuracy, as well as protocol development for DNA extraction (Vaillancourt and Buell, 2019;Vilanova et al, 2020), have facilitated the analysis of crop pangenomes (Alonge et al, 2020;Liu et al, 2020;Qin et al, 2021), and the development of near-complete human and Arabidopsis genome sequences (Nurk et al, 2021;Naish et al, 2021).…”