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- Title
Culicidae-centric metabarcoding through targeted use of D2 ribosomal DNA primers.
- Authors
Pedro, Pedro M.; Amorim, Jandui; Rojas, Martha V. R.; Sá, Ivy Luizi; Galardo, Allan Kardec Ribeiro; Santos Neto, Noel Fernandes; de Carvalho, Dario Pires; Ribeiro, Kaio Augusto Nabas; Razzolini, Maria Tereza Pepe; Mureb Sallum, Maria Anice
- Abstract
A practical limitation to many metabarcoding initiatives is that sampling methods tend to collect many non-target taxa, which become "amplicon noise" that can saturate Next Generation Sequencing results and lead to both financial and resource inefficiencies. An available molecular tool that can significantly decrease these non-target amplicons and decrease the need for pre-DNA-extraction sorting of bycatch is the design of PCR primers tailored to the taxa under investigation. We assessed whether the D2 extension segment of the 28S ribosomal operon can limit this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude from amplification most other taxa likely to be collected with current sampling apparatuses.Weshow that, given enough sequencing depth,D2is an effective marker for the detection of mosquito sequences within mock genomicDNA pools. As few as 3,050 quality-filtered Illumina reads were able to recover all 17 species in a bulk pool containing as little as 0.2% of constituent DNA from single taxa. We also mixed these mosquito DNA pools with high concentrations of non-Culicidae bycatch DNA and show that the component mosquito species are generally still recoverable and faithful to their original relative frequencies. Finally, we show that there is little loss of fidelity in abundance parameters when pools from degraded DNA samples were sequenced using the D2 primers.
- Subjects
DNA primers; RIBOSOMAL DNA; SPECIES pools; GENETIC barcoding; MOSQUITOES; DNA
- Publication
PeerJ, 2020, p1
- ISSN
2167-8359
- Publication type
Article
- DOI
10.7717/peerj.9057