Department of Civil and Environmental Engineering, Duke University, 127 Hudson Hall, Box 90287, Durham, NC 27708, USA.
Department of Civil and Environmental Engineering, Washington State University, 405 Spokane street, Sloan 101, Box 642910, Pullman, WA 99164, USA.
FEMS Microbiol Ecol. 2020 Jul 1;96(7). doi: 10.1093/femsec/fiaa110.
Due to the sequence homology between the bacterial 16S rRNA gene and plant chloroplast and mitochondrial DNA, the taxonomic characterization of plant microbiome using amplicon-based high throughput sequencing often results in the overwhelming presence of plant-affiliated reads, preventing the thorough description of plant-associated microbial communities. In this work we developed a PCR blocking primer assay targeting the taxonomically informative V5-V6 region of the 16S rRNA gene in order to reduce plant DNA co-amplification, and increase diversity coverage of associated prokaryotic communities. Evaluation of our assay on the characterization of the prokaryotic endophytic communities of Zea mays, Pinus taeda and Spartina alternifora leaves led to significantly reducing the proportion of plant reads, yielded 20 times more prokaryotic reads and tripled the number of detected OTUs compared to a commonly used V5-V6 PCR protocol. To expand the application of our PCR-clamping assay across a wider taxonomic spectrum of plant hosts, we additionally provide an alignment of chloroplast and mitochondrial DNA sequences encompassing more than 200 terrestrial plant families as a supporting tool for customizing our blocking primers.
由于细菌 16S rRNA 基因与植物叶绿体和线粒体 DNA 之间存在序列同源性，因此使用基于扩增子的高通量测序对植物微生物组进行分类特征描述时，通常会导致植物相关阅读的压倒性存在，从而无法彻底描述与植物相关的微生物群落。在这项工作中，我们开发了一种 PCR 阻断引物检测方法，针对 16S rRNA 基因的分类信息丰富的 V5-V6 区域，以减少植物 DNA 的共扩增，并增加相关原核群落的多样性覆盖范围。我们的检测方法用于表征玉米、火炬松和互花米草叶片中的原核内生群落，结果显著降低了植物阅读的比例，与常用的 V5-V6 PCR 方案相比，产生了 20 倍的原核阅读量和三倍的检测 OTU 数量。为了将我们的 PCR 阻断检测方法扩展到更广泛的植物宿主分类范围，我们还提供了一个包含 200 多个陆地植物科的叶绿体和线粒体 DNA 序列比对，作为定制我们的阻断引物的支持工具。
