Systematic Comparisons between Long-Read- and Short-Read-Based Amplicon Sequencing to Characterize Mixed Microalgal Communities

Abstract The 18S rRNA gene has emerged as the primary molecular marker for amplicon-based characterization of microalgal communities, including in wastewater treatment systems, yet trade-offs between short- and long-read approaches remain poorly defined. We systematically compared V8–V9 short-read sequencing (SR; Illumina MiSeq), near-full-length long-read sequencing with ss5ss3 primers (LR; PacBio Sequel II), and computationally extracted V8–V9 regions from LR data (LS). In silico evaluation of multiple primer pairs revealed differences in predicted taxonomic recovery, while incomplete reference sequences and taxonomic misannotations complicated performance assessments. In environmental samples, SR and LR captured different aspects of sequence diversity: SR recovered a broader set of sequence variants, whereas the longer LR amplicons retained greater sequence information and discrimination. Truncating LR sequences to V8–V9 collapses distinct ASVs. The taxonomic advantage of LR was classification-workflow-dependent and was particularly apparent at genus and species levels with the eukaryote-focused workflow. Differential-abundance analysis identified species-level Chlorophyta associations in LR, with consistent higher-level taxonomic patterns across periods, whereas SR additionally revealed largely period-specific associations with heterotrophic protists. Despite its finer taxonomic resolution, LR did not provide a clearer whole-community distinction between process performance states (i.e., phosphorus-removal, emphasizing that the sequencing approach should be selected according to the information required.