A full‐length 18S ribosomal DNA metabarcoding approach for determining protist community diversity using Nanopore sequencing

Abstract

Protist diversity studies are frequently conducted using DNA metabarcoding methods. Currently, most studies have utilized short read sequences to assess protist diversity. One limitation of using short read sequences is the low resolution of the markers. For better taxonomic resolution longer sequences of the 18S rDNA are required because the full‐length has both conserved and hypervariable regions. In this study, a new primer pair combination was used to amplify the full‐length 18S rDNA and its efficacy was validated with a test community and then validated with field samples. Full‐length sequences obtained with the Nanopore MinION for protist diversity from field samples were compared with Illumina MiSeq V4 and V8‐V9 short reads. Sequences generated from the high‐throughput sequencers are Amplicon Sequence Variants (ASVs). Metabarcoding results show high congruency among the long reads and short reads in taxonomic annotation at the major taxonomic group level; however, not all taxa could be successfully detected from sequences. Based on the criteria of ≥95% similarity and ≥1000 bp query length, 298 genera were identified by all markers in the field samples, 250 (84%) were detected by 18S, while only 226 (76%) by V4 and 213 (71%) by V8‐V9. Of the total 85 dinoflagellate genera observed, 19 genera were not defined by 18S dinoflagellate ASVs compared to only three among the total 52 diatom genera. The discrepancy in this resolution is due to the lack of taxonomically available 18S reference sequences in particular for dinoflagellates. Overall, this preliminary investigation demonstrates that application of the full‐length 18S rDNA approach can be successful in field studies.

The Ribosomal Operon Database (ROD): A full-length rDNA operon database derived from genome assemblies

“…In contrast to short-read DNA metabarcoding, long-read sequencing technologies make it possible to sequence longer stretches of the rDNA region (Jamy et al, 2020), even the entire rDNA operon (Gaonkar & Campbell, 2024; Hooper et al, 2023; Wurzbacher et al, 2019), with high precision (Callahan et al, 2019). This calls for new reference sequences covering the whole rDNA operon.…”