1. SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species
- Author
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Universitat Politècnica de València. Instituto Universitario Mixto de Biología Molecular y Celular de Plantas - Institut Universitari Mixt de Biologia Molecular i Cel·lular de Plantes, Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia, Universitat Politècnica de València. Instituto Universitario de Conservación y Mejora de la Agrodiversidad Valenciana - Institut Universitari de Conservació i Millora de l'Agrodiversitat Valenciana, Generalitat Valenciana, COMISION DE LAS COMUNIDADES EUROPEA, Universitat Politècnica de València, Japan Society for the Promotion of Science, Ministerio de Educación, Cultura y Deporte, Vilanova Navarro, Santiago, Alonso-Martín, David, Gramazio, Pietro, Plazas Ávila, María de la O, García-Fortea, Edgar, Ferrante, Paola, Schmidt, Maximilian, Díez Niclós, Mª José Teresa De Jesús, Usadel, Björn, Giuliano, Giovanni, Prohens Tomás, Jaime, Universitat Politècnica de València. Instituto Universitario Mixto de Biología Molecular y Celular de Plantas - Institut Universitari Mixt de Biologia Molecular i Cel·lular de Plantes, Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia, Universitat Politècnica de València. Instituto Universitario de Conservación y Mejora de la Agrodiversidad Valenciana - Institut Universitari de Conservació i Millora de l'Agrodiversitat Valenciana, Generalitat Valenciana, COMISION DE LAS COMUNIDADES EUROPEA, Universitat Politècnica de València, Japan Society for the Promotion of Science, Ministerio de Educación, Cultura y Deporte, Vilanova Navarro, Santiago, Alonso-Martín, David, Gramazio, Pietro, Plazas Ávila, María de la O, García-Fortea, Edgar, Ferrante, Paola, Schmidt, Maximilian, Díez Niclós, Mª José Teresa De Jesús, Usadel, Björn, Giuliano, Giovanni, and Prohens Tomás, Jaime
- Abstract
[EN] 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 euro/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 fromSolanum elaeagnifoliumusing 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 inexpensiv
- Published
- 2020