Evaluating individual and pooled mosquito samples for virome analysis using PacBio HiFi long-read metagenomics

Background: Characterizing the mosquito virome is essential for understanding host-microbiota interactions and vector competence. Although sample scale (individual vs pools) and sequencing approach (short- vs long-reads approaches) are of key importance in the design of a mosquito virome study, technical limitations may affect the choice about these issues. In the present study, using larvae and adults of the sea-rock pool mosquito Aedes mariae, we propose a methodological framework based on PacBio HiFi long-read technology. This approach combines extended read length with high base-level accuracy (>99.9%), enabling improved species-level taxonomic resolution. To evaluate how sampling scale influences library robustness and virome resolution, we analysed single individuals and pools of increasing size of larval and adult stages.Results: Eight samples, including single individuals and pools of increasing size for both Ae. mariae larvae and adults, were analysed. Although total DNA and RNA concentrations increased with biomass, removal of host and free nucleic acids reduced viral material below detection limits, requiring an amplification step. Sequence-Independent Single-Primer Amplification (SISPA) yielded quantifiable viral DNA and cDNA across all samples, with marked gains between one and three individuals and a tendency to stabilise as pool size increased. A single individual provided sufficient material for library construction, while pools of three individuals yielded more stable outputs. Amplicons were highly homogeneous (1055-1222 bp), and HiFi reads showed high quality (Q34-Q35). Sequencing output did not increase linearly with pool size; however, pools of three individuals exhibited the highest proportions of assigned viral reads (larvae circa 38%, adults circa 34%), whereas unassigned reads predominated in samples with fewer individuals, reaching up to circa 80% in larvae and circa 99% in single adults.Conclusions:The results confirm the robustness of the PacBio HiFi long-read metagenomic workflow for mosquito virome characterization. The method is effective for both single specimens and pooled samples: single-individual analyses enable exploration of individual variability and rare or previously undescribed viruses, while pooling better captures the shared viral diversity at the population level.