Culicidae-centric metabarcoding through targeted use of ribosomal DNA primers

A practical limitation to many metabarcoding initiatives is that sampling methods tend collect many non-target taxa, which become “amplicon noise” that can saturate NGS sequencing results and lead to both financial and resource inefficiencies. We assess whether the D2 extension segment of the 28S ribosomal operon can decrease this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude most other taxa likely to be collected with current sampling apparatuses. We show that, given enough sequencing depth, D2 is an effective marker for the detection of mosquito sequences within mock genomic DNA pools.