Ecological Indicators ( IF 6.9 ) Pub Date : 2020-10-08 , DOI: 10.1016/j.ecolind.2020.106973 Joseph Sevigny , Francesca Leasi , Stephen Simpson , Maikon Di Domenico , Katharina M. Jörger , Jon L. Norenburg , W. Kelley Thomas
High-throughput DNA sequencing studies allow rapid and widescale investigations into complex assemblages of organisms that would otherwise remain infeasible. Recently scientists have begun to apply metagenomic sequencing (sequencing of the complete pool of DNA from a sample) to investigate microscopic animal communities. Mitochondrial genomes are an important locus for such studies; however, current databases of complete mitochondrial genomes remain incomplete, and many important meiofaunal clades are underrepresented or missing entirely. This underrepresentation is likely due to the low amount of input DNA acquired from meiofaunal individuals, the impossibility of pooling animals because of high levels of cryptic speciation, and the paucity of researchers collecting and studying these animals. Here we help to alleviate the first two barriers by developing a method of enriching animal mtDNA from next-generation sequencing libraries by using hybridization capture probes. These hybrid-capture probes were designed based on the complete collection of mitochondrial genomes available from GenBank and are suitable for enrichment of diverse animal lineages. We demonstrate the protocol on forty-seven meiofaunal specimens spanning four different phyla. Overall, we observed an average of ~450× fold enrichment over standard NGS sequencing library preparation. In addition, we were able to enrich the mtDNA from taxa distantly related to those in the database, indicating that even animals that were not directly targeted by the probe design can be enriched with this method.
中文翻译:
杂交捕获探针对后生线粒体DNA的靶富集
高通量DNA测序研究可以对大规模复杂的生物体进行快速而广泛的研究,否则这些生物体将不可行。最近,科学家已开始应用宏基因组测序(从样品中提取完整的DNA序列)来研究微观动物群落。线粒体基因组是此类研究的重要场所。然而,目前完整的线粒体基因组数据库仍然不完整,许多重要的系膜进化枝的代表性不足或完全缺失。这种表示不足的原因可能是由于从动植物个体获得的输入DNA数量少,由于高水平的隐性物种形成而无法汇集动物,以及缺乏收集和研究这些动物的研究人员。在这里,我们通过开发一种使用杂交捕获探针从下一代测序文库中富集动物mtDNA的方法,来帮助缓解前两个障碍。这些杂交捕获探针是基于可从GenBank获得的线粒体基因组的完整集合而设计的,适用于丰富各种动物谱系。我们展示了跨越四个不同门的47个半瓣标本的协议。总体而言,我们观察到比标准NGS测序文库制备平均富集约450倍。此外,我们能够从与数据库中远距离相关的类群中富集mtDNA,这表明即使不是直接由探针设计靶向的动物也可以使用此方法进行富集。