Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes
Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes
复制标题
DOI：
10.1371/journal.pone.0209499
复制
发表时间：
2019-02
期刊：
影响因子：
3.7
通讯作者：
Stephen M. Richards;N. Hovhannisyan;Matthew Gilliham;Joshua Ingram;B. Skadhauge;Holly Heiniger;B. Llamas
中科院分区：
文献类型：
--
作者：
Stephen M. Richards;N. Hovhannisyan;Matthew Gilliham;Joshua Ingram;B. Skadhauge;Holly Heiniger;B. Llamas
在高通量测序（HTS）之前，使用溶液中寡核苷酸探针的杂交捕获已迅速成为从降解或古老样品富集特定DNA基因座的优选方法。几家公司合成了用于溶液中杂交捕获的探针组，但是这些商业试剂通常是昂贵的。已经描述了用于经济的内部探针合成的方法，但是它们没有直接解决商业合成的探针的主要优点之一：匹配许多物种的探针序列可以平行合成并合并。制造“生物遗传多样性”探针的能力提高了商业探针组的成本效益，因为它们可以用于多个项目（或涉及多个物种的项目）。然而，用内部方法复制这种方法是劳动密集型的，因为必须首先为每个感兴趣的物种生成模板分子。虽然已经观察到探针可以用于富集遗传学上远距离的靶标，但是尚未测试这种效应补偿内部合成的探针组中遗传学上多样性探针的缺乏的能力。在这项研究中，我们提出了一个完善的协议，在内部的RNA探针合成和评估的能力，使用这种方法从一个单一的物种，成功地丰富的目标基因座在遗传学上遥远的物种。我们证明了使用来自胎盘哺乳动物野牛（野牛属）的长距离PCR产物合成的探针。可用于富集鸟类和有袋动物（但不是植物）的线粒体DNA。重要的是，我们的结果是以类似市售试剂的大约三分之一的成本获得的。
Hybridization capture with in-solution oligonucleotide probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make “phylogenetically diverse” probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents.