Abstract
BackgroundThe 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.ResultsSILEX 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.ConclusionsA 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.
Highlights
The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information
We present a novel, fast and inexpensive DNA extraction protocol that combines the advantages of CTAB-based extraction coupled with a purification on a silica matrix
The DNA obtained was successfully used to construct long insert size Nanopore libraries for a de novo genome assembly, which can be difficult for recalcitrant species [40], proving its suitability for third-generation sequencing platforms. We demonstrate that this new method combines the advantages of commercial kits with those of a CTAB-based method being suitable for routinely DNA screening and next generation sequencing (NGS) platforms
Summary
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 plat‐ forms. The CTAB DNA extraction protocol developed by Doyle and Doyle [10] is one of the most widely used by plant researchers Several modifications of this protocol have been implemented in order to minimize contamination by other compounds of specific tissues of species [7, 11, 12]. These modifications, apart from being species or tissue-specific and frequently not removing completely interfering compounds, are time-consuming due to many handling steps, and are not suitable for highthroughput applications [13, 14]
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