High-throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long-read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500-bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high-quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

中文翻译：

---

真核rDNA操纵子的长期metabarcoding系统发育和分类学解决环境多样性。

扩增子大小低于500 bp的高通量DNA metabarcoding彻底改变了环境微生物多样性的分析方法。但是，这些短区域包含有限的系统发育信号，这使得在完整的系统发育推断中使用环境DNA不切实际。短扩增子的这种较小的系统发育分辨率可以通过新的长读测序技术来克服。为了验证该想法，我们扩增了土壤DNA并使用了PacBio环形共有序列（CCS）获得了一个大约4500 bp的区域，该区域跨越了大多数真核生物小亚基（18S）和大亚基（28S）核糖体DNA基因。我们首先使用新颖的策展工作流程处理CCS读物，生成了650个包含物理链接的18S和28S区域的高质量操作分类单位（OTU）。要将分类法分配给这些OTU，我们开发了一种基于18S区域的系统发育感知方法，与基于相似性的方法相比，该方法显示出更高的准确性和灵敏度。然后，将经过分类学注释的OTU与可用的18S和28S参考序列进行组合，以推断出涵盖所有主要真核生物类的已完全分辨的系统发育史，从而使我们能够准确地得出环境多样性的进化起源。总共包括1,019个序列，其中大多数（58％）对应于新的长环境OTU。长阅读还使我们能够直接研究环境序列本身之间的关系，这相对于将短阅读放置在参考系统发生学上而言，是一个关键优势。一起，