Journal Article

FZJ-2020-02843

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png SILEX: a fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

Vilanova, S. (Corresponding author) ; Alonso, D. ; Gramazio, P. ; Plazas, M. ; García-Fortea, E. ; Ferrante, P. ; Schmidt, M.FZJ*RWTH* ; Díez, M. J. ; Usadel, B.FZJ* ; Giuliano, G. ; Prohens, J.

2020
BioMed Central London

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Please use a persistent id in citations: http://hdl.handle.net/2128/25653  doi:10.1186/s13007-020-00652-y

Abstract: Background: The use of sequencing and genotyping platforms has undergone dramatic improvements, enablingthe generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomicDNA (gDNA) in sufcient concentrations is often a main limitation, especially for third-generation sequencing plat‑forms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly andfrequently 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 speciesand tissues.Results: SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silicamatrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitorycompounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kitin a variety of species, including recalcitrant ones, from diferent families. In comparison with the other methods, SILEXyielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 minby one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtainedwith 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 thisprotocol was assessed by Pulsed-feld gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencingplatforms 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 awide variety of plants and tissues. SILEX ofers an easy, scalable, efcient and inexpensive way to extract DNA for vari‑ous next-generation sequencing applications including SPET and Nanopore among others.Keywords: DNA extraction, CTAB protocol, Silica matrix, Contaminant-free DNA, High-molecular-weight DNA, Nextgeneration sequencing, High-throughput genotyping, Recalcitrant species, SPET, Nanopore

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Contributing Institute(s):

1. Bioinformatik (IBG-4)

Research Program(s):

1. 582 - Plant Science (POF3-582) (POF3-582)
2. 583 - Innovative Synergisms (POF3-583) (POF3-583)

Appears in the scientific report 2020

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