Proper fin-clip sample collection for molecular analyses in the age of eDNA

Environmental DNA (eDNA), the collection of trace amounts of DNA from water for species detection, is a new molecular technique that is rapidly increasing in usage in fisheries biology. eDNA most commonly uses species-specific primers and probes in quantitative PCR (qPCR) to amplify eDNA from target species, while excluding amplification of eDNA from non-target species, thus allowing researchers to determine if a target species is present in a given water body (Goldberg et al., 2015; McKelvey et al., 2016). When designing and testing eDNA assays for qPCR, researchers use tissue derived DNA, usually from fin-clips, of both target and non-target species to validate species-specificity in the laboratory (Wilcox et al., 2013). eDNA assays target mitochondrial DNA because mitochondrial DNA has a very high copy number per cell; each cell contains hundreds to thousands of copies of the mitochondrial genome. qPCR is a remarkably sensitive technique capable of detecting just one copy of target DNA. Tissue DNA extracts contain very high concentrations of DNA, generally many orders of magnitude higher than would be found in an eDNA sample. For this reason, cross-contamination between target-species and non-target species samples during fin clipping is a major risk. Such cross-contamination not only makes fin-clip samples unusable for validation of eDNA assays, it can also waste limited time and resources in the assay validation process. Thus, researchers are urged to collect fin-clip samples for molecular analyses or museum collections in a manner that is compatible with their use in future eDNA assay development.

Cross-contamination of fin-clip samples occurs when collectors use the same instrument for fin clipping of multiple species during the same sampling event without sterilizing the instrument between samples. If just a few cells, or even portions of cells are left behind on the instrument, this is more than enough to cause cross-contamination. Such low level cross-contamination is typically not of concern for other common types of molecular analyses such as DNA sequencing or microsatellite analyses, because DNA from the fin clip itself is much more abundant than contaminating DNA from previous samples. This is not the case for species-specific qPCR due to its high sensitivity. To reduce cross-contamination, collectors should use sterile clipping instruments for each sample, and should use a new pair of disposable latex gloves when handling each fish. Instruments can be sterilized by soaking in 10% bleach solution for 15 min. Ten percent bleach is effective in degrading DNA so that it cannot be amplified by PCR. However it is important that the bleach used is fresh, and new 10% bleach solution should be made up for each sterilization, as oxidation quickly reduces the effectiveness of bleach. Because such sterilization is not practical to perform in the field between each sample, bringing enough pre-sterilized instruments for each sample, or using sterile, disposable scalpels for fin-clipping may be desirable.

Laboratory researchers should also consider the source of fin clip samples when designing and validating eDNA assays. When using existing collections, care should be taken to select non-target samples for eDNA assay validation that were not collected immediately after, or even in the same collection trip as target species. Cross-contamination has been observed in many museum collection samples (Rodgers et al., 2017), usually where samples were collected immediately after the target species, but in some cases where samples were collected up to several days after the target species. Cross contamination can be distinguished from cross-amplification by sequencing qPCR amplicons if sequence polymorphisms exist between target and non-target species in the internal assay sequence. If sequences derived from amplification of non-target samples match the target species sequence, this indicates that the non-target sample is cross-contaminated with target species DNA. If non-target species samples collected independently of target species samples are not available, surface sterilization of samples with bleach prior to DNA extraction may reduce cross-contamination. Low-level contamination, however, may still be observed, even after surface sterilization. If samples are surfaced sterilized with bleach, care should be taken to wash of all bleach residue prior to extraction, as bleach can act as a strong PCR inhibitor.

Because eDNA is a recent technique, collectors have not all foreseen the need to take extreme care to avoid cross-contamination when collecting fin-clip samples for molecular analyses in the past. As eDNA is rapidly increasing, and will continue to increase as a valuable tool for fisheries biology, collectors can better facilitate future eDNA research by adopting sterile protocols for collecting fin-clip samples for molecular analyses so that these samples can be used in the validation of eDNA assays. Sterile protocols are not difficult to implement in the field, and will increase the value of sample collections for future eDNA research.