Evaluating the Effect of Sampling Scale on Mosquito Virome Characterization Using PacBio HiFi Long-Read Metagenomics

Area of Science:

• Vector biology
• Virology
• Metagenomics

Background:

• Characterizing mosquito viromes is crucial for understanding vector competence and host-microbiota interactions.
• Virome analysis can be affected by sampling scale, sequencing methods, and host genome depletion.

Purpose of the Study:

• To evaluate the impact of sampling scale on mosquito virome characterization using third-generation sequencing (TGS).
• To compare single-individual versus pooled mosquito sample analyses before and after host genome removal.

Main Methods:

• Utilized PacBio HiFi long-read TGS metagenomics on L4 larvae and adults of *Aedes mariae*.
• Analyzed single individuals and pools of increasing size.
• Performed host genome removal to assess its effect on viral read composition.

Main Results:

• Sequencing yield was not proportional to pool size.
• Host genome removal decreased total reads but enriched for viral reads.
• Virome diversity and taxonomic richness increased with pool size, reflecting individual contributions.
• A significant proportion of unassigned reads indicates the presence of novel viruses.

Conclusions:

• Both single-individual and pooled sample analyses are feasible and provide complementary insights.
• Pooled samples enhance detection of broader viral diversity and shared viral components.
• Single-individual analyses are valuable for identifying low-abundance viral taxa and individual-specific viromes.