■测序和基因分型平台的使用经历了巨大的改进,能够产生丰富的基因组信息。尽管取得了这些进展,足够浓度的高质量基因组DNA(gDNA)的可用性通常是主要限制,特别是第三代测序平台。各种DNA提取方法和商业试剂盒是可用的。然而,其中许多是昂贵的,并且经常产生低产量或低质量的DNA,不适合下一代测序(NGS)平台。这里,我们描述了一种快速且廉价的DNA提取方法(SILEX),适用于多种植物物种和组织。
■SILEX是一种高通量DNA提取方案,基于标准CTAB方法与DNA二氧化硅基质回收,这允许获得不含抑制性化合物的NGS质量高分子量基因组植物DNA。将SILEX与标准CTAB提取方案和各种物种中常见的商业提取试剂盒进行了比较,包括顽固的,来自不同的家庭。与其他方法相比,SILEX产生更高浓度和更高质量的DNA。一个人可以在96分钟内手动提取48个样品,费用为0.12€/样品试剂和耗材。使用IlluminaHiSeq2500平台,使用单引物富集技术(SPET)成功对使用SILEX或商业试剂盒获得的数百个番茄gDNA样品进行了基因分型。此外,使用该方案从茄子提取的DNA通过脉冲场凝胶电泳(PFGE)进行评估,为大多数需要高分子量DNA的测序平台(如Nanopore或PacBio)获得合适的大小范围。
■高吞吐量,开发了快速且廉价的DNA提取方案,并对各种植物和组织进行了验证。SILEX提供了一个简单的,可扩展,高效和廉价的方式来提取DNA的各种下一代测序应用,包括SPET和Nanopore等。
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.