Comparison of five extraction protocols and direct PCR for the recovery of trace DNA in chironomid pupal exuviae

Background: Efficient DNA extraction is critical to the success of species identification using DNA barcoding and metabarcoding, especially when the total amount of extracted DNA is expected to be low. Here, we compare the performance of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Insecta: Diptera) is a group of species-rich aquatic macroinvertebrates widely distributed in freshwater environments and considered valuable bioindicators of water quality. A commonly used form of sampling chironomids involves collection of pupal exuviae. Thus, using DNA barcoding to identify chironomid pupal exuviae can be an important asset to understanding freshwater ecology as well as advancing chironomid taxonomy through life stage associations. Results: Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. Several of the extracts were contaminated with DNA from non-target organisms; only 13.7% of the sequences produced from these extracts matched chironomid sequences. There were significant differences in the extraction methods and direct PCR with regards to cost, handling time,DNAquantity, PCR success, sequence success, and the ability to sequence target taxa. The NucleoSpin Tissue XS Kit, DNeasy Blood and Tissue kit, and QuickExtract DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. Significance: The observed differences in DNA extraction protocol performance on trace DNA should be of interest to studies focusing on noninvasive sampling in aquatic environments, such as environmental barcoding and metabarcoding.