Klure Dylan M, Greenhalgh Robert, Dearing M Denise
University of Utah, School of Biological Sciences, 257 S 1400 E Rm 201, Salt Lake City, UT, 84112.
Mamm Res. 2022 Oct;67(4):499-509. doi: 10.1007/s13364-022-00646-2. Epub 2022 Jul 30.
High-throughput sequencing approaches have revolutionized how we study animal diets by enabling the detection of dietary components from the metabarcoding of DNA in excrement. Mitochondrial cytochrome oxidase C subunit I (mtCOI) DNA metabarcoding is commonly used to study the diets of arthropod-feeding animals; however, this approach is susceptible to nontarget amplification of the consumer species mtCOI locus. Nontarget amplification is often an unforeseen complication that can drastically reduce the quality and utility of the results generated by high-throughput amplicon sequencing. By interrogating the diets of new world rodents in the genus (woodrats) in both natural and captive settings, we demonstrate that nontarget amplification can drastically reduce the total read abundance of detected arthropod taxa in fecal samples and inhibit downstream analyses of dietary diversity and composition metrics. Using the results from these investigations, we offer a guide on how to identify concerns for nontarget amplification when selecting degenerate primers for DNA metabarcoding studies and recommend several approaches that can reduce or eliminate nontarget amplification. Lastly, for the community interested in investigating the diets of arthropod-feeding rodents, we generated a database containing the degree of mismatch between publicly available Rodentia mtCOI sequences and four common universal mtCOI primer sets to be used as a resource for inferring the relative risk of nontarget amplification when designing arthropod metabarcoding studies in rodent systems. This guide will be especially useful for researchers working with consumer species that have not previously been studied.
高通量测序方法彻底改变了我们研究动物饮食的方式，它能够通过对粪便中的DNA进行代谢条形码分析来检测饮食成分。线粒体细胞色素氧化酶C亚基I（mtCOI）DNA代谢条形码分析常用于研究以节肢动物为食的动物的饮食；然而，这种方法容易出现目标物种mtCOI基因座的非靶向扩增。非靶向扩增通常是一种意想不到的复杂情况，会大幅降低高通量扩增子测序产生的结果的质量和实用性。通过研究自然和圈养环境中新世界 属（林鼠）啮齿动物的饮食，我们证明非靶向扩增会大幅降低粪便样本中检测到的节肢动物类群的总读数丰度，并抑制对饮食多样性和组成指标的下游分析。利用这些调查结果，我们提供了一份指南，介绍在为DNA代谢条形码研究选择简并引物时如何识别非靶向扩增问题，并推荐了几种可以减少或消除非靶向扩增的方法。最后，对于有兴趣研究以节肢动物为食的啮齿动物饮食的群体，我们生成了一个数据库，其中包含公开可用的啮齿动物mtCOI序列与四种常见通用mtCOI引物集之间的错配程度，用作推断在啮齿动物系统中设计节肢动物代谢条形码研究时非靶向扩增相对风险的资源。本指南对于研究以前未研究过的目标物种的研究人员尤其有用。
