Biodiversity Assessment of Insect from Environmental Samples Using qPCR and Next-Generation Parallelized Sequencing of DNA Barcodes
This thesis employs three bioindicator species of mayfly (Insecta: Ephemeroptera) and three of caddisfly (Insecta: Trichoptera) as models to develop a reliable biodiversity and biomonitoring assessment approach by using quantitative PCR (qPCR) and next generation sequencing (NGS) technology. Quantitative PCR was employed to assess the efficiency of species-specific PCR primers in amplifying their target species versus other taxa from closely or distantly related taxonomic groups from benthic habitats. Results showed qPCR can be used as a practical test for evaluating PCR primers for amplifying specific taxa in mixed environmental samples although it might be influenced by amplification bias. Target specific primers are an alternate to presumably universal primers. Each primer set can be tested and optimized using qPCR prior to use in next-generation sequencing. qPCR results showed corroboration with 454 pyrosequence data and hence it can be used in experimental design procedure for NGS based biomonitoring which could indicate that qPCR is a useful tool for selecting primers in the NGS amplicon preparation.