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

Abstract

High-throughput environmental DNA metabarcoding has revolutionized the analysis of microbial diversity, but this approach is generally restricted to amplicon sizes below 500 base pairs. These short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. However, new long-read sequencing technologies such as the Pacific Biosciences platform may provide sufficiently large sequence lengths to overcome the poor phylogenetic resolution of short amplicons. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain a ~4500 bp region of the eukaryotic rDNA operon spanning most of the small (18S) and large subunit (28S) ribosomal RNA genes. The CCS reads were first treated with a novel curation workflow that generated 650 high-quality OTUs containing the physically linked 18S and 28S regions of the long amplicons. In order 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 and phylogenetic placement-based methods using shorter reads. 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 to accurately derive the evolutionary origin of environmental diversity.A total of 1019 sequences were included, of which a majority (58%) corresponded to the new long environmental CCS reads. Comparisons to the 18S-only region of our amplicons revealed that the combined 18S-28S genes globally increased the phylogenetic resolution, recovering specific groupings otherwise missing. The long-reads also allowed to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Altogether, 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.

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens

“…Both Oxford Nanopore and PacBio sequencing platforms are rapidly evolving in terms of read length and base-calling accuracy. At the moment, Sequel seems to be the best choice for metabarcoding of regular-size (600 to 1,000 bp) and long (up to 4.5 kb) amplicons (6,7,12,(56)(57)(58) and for barcoding using ultralong markers (up to 7 kb) (59). The recently released M8 chemistry has reduced the per read cost of PacBio sequencing roughly threefold.…”

“…Although we could reach up to 99.5% accuracy with Ͼ100 MinION reads, 100 reads were considered sufficient for generating principally error-free barcodes for animals using the 1D 2 approach (30). For PacBio instruments, a single read may be enough for reads of around 2,000 bases, but three or more may be needed for longer fragments and to average over PCR errors (58).…”

Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies: Current Uses and Future Directions

“…A better, though not portable, alternative for metabarcoding may be offered by Pacific Biosciences. The circular sequencing offered by Pacific Biosciences’ Sequel considerably reduces the raw read error, making this a promising technology for metabarcoding applications [25,26]. A similar technique is not currently available for nanopore sequencing.…”

“…At the same time, targeting particular amplicons in the genome makes it possible to minimize sequencing effort compared to genome skimming applications. With appropriate error correction applied, the method may also be suitable to generate long-read metabarcoding data [25,26].…”

Intra‐genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing

“…New sequencing technologies have been developed with the ability of high‐throughput sequencing longer nucleotide fragments in real‐time, such as Oxford Nanopore Technologies (ONT) or Pacific Bioscience (PacBio). These sequencing methods have already showed their useful capabilities, for example PacBio has developed a circular consensus sequencing resulting in near‐zero error long reads, improving aspects such as genome assembly (Wenger et al ., 2019) or even phylogenetic analysis of environmental diversity (Jamy et al ., 2019). However, its limited high‐throughput sequencing ability and its relatively high cost (Goodwin et al ., 2016) may affect the sequencing depth at a metabarcoding community level.…”