Systematic comparisons between long-read and short-read based amplicon sequencing to characterize mixed microalgal communities.

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 V8V9 short-read sequencing (Illumina MiSeq), full-length long-read sequencing with ss5ss3 primers (PacBio Sequel II), and computationally extracted V8V9 regions from long-read data. Both in silico and in vitro analyses confirmed V8V9 captured broader taxonomic coverage than ss5ss3, though partial reference sequences and taxonomic misannotations biased in silico assessments. Long reads taxonomic advantage was database-dependent, constrained by SILVA databases genus-level curation but fully realized when paired with the species-level-curated and eukaryotes-focused PR2 (Protist Ribosomal Reference) database. Long-read sequencing uniquely identified amplicon sequence variants (ASVs) assigned to key phosphorus assimilators (Scenedesmus obliquus, Desmodesmus sp., and Acutodesmus sp.) at the species level during successful phosphorus removal in a full-scale microalgal cultivation system, while V8V9 short-read sequencing revealed ASVs assigned to algal-predatory (Leptophryidae) and bacterivorous (Choanoflagellata and Rhogostoma-lineage) protists when performance declined, suggesting grazing pressure on the phosphorus-removing community. Although both approaches performed comparably for operational monitoring, these complementary strengths support short-read sequencing for routine community profiling and long-read sequencing for detailed functional investigations of Chlorophyta.