Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

Abstract

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.

Perspectives from Ten Years of Protist Studies by High‐Throughput Metabarcoding

“…Most recently, Jamy et al. () analyzed soil protists by producing sequences ~4,500 bp long using broad eukaryotic primers that target a region spanning from 18S to 28S rRNA genes. Taking advantage of this increased phylogenetic signal, it is possible to develop a phylogeny‐aware method for taxonomic annotation.…”

“…Another benefit of long‐amplicon sequencing, as mentioned above, is that we can produce reference phylogenies including not only reference sequences but also newly obtained environmental sequences (Jamy et al. ). This means that denser and more robust reference trees will become available, which, we believe, will be especially powerful for annotation of shorter Illumina sequences.…”

Polyphyletic origin, intracellular invasion, and meiotic genes in the putatively asexual agamococcidians (Apicomplexa incertae sedis)

“…cyamus and Rh. polygordiae form long tree branches which may cause the long branch attraction artifact, and the earlier studies used SSU rDNA phylogenies only, which have inferior resolution to the whole rRNA operon [24][25][26][27] .…”

Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

“…The technical limitations of short-read platforms have recently been tested using long-read sequencing platforms, namely Pacific Biosciences (PacBio) and Oxford Nanopore. These platforms can sequence amplicons of the complete 16S rRNA gene [19][20][21][22] and even the entire rRNA gene operon, including the ITS and small and large subunit rRNA genes (>4000 bp), of prokaryotes [23][24][25] and microbial eukaryotes [26][27][28]. A major drawback of long-read sequencing is its higher perbase error rate compared to short-read sequencing.…”