Alligator Snapping Turtle eDNA Analysis from Filtered Water Samples

The proposed work is to analyze samples for a single species, the Alligator Snapping Turtle, for environmental DNA (eDNA). Water samples (135 total) were collected from lakes and streams and filtered in the laboratory using analytical filter funnels containing 0.45um filters. Filters, containing eDNA, were removed from filter funnels and placed in labeled microcentrifuge tubes and the tubes were placed in a deep freeze. Each microcentrifuge tube contains 2 filters from the same water sample.

Samples will be shipped in an insulated container, containing cold packs, to a laboratory for analysis. Each tube should be considered as an independent sample. Laboratory is required to detect sequences of Alligator Snapping Turtle DNA from filter medium and ensure no cross contamination of samples. Laboratory must provide a detailed report showing CT scores for each run (3 runs per sample) and if each sample has a positive or negative result for Alligator Snapping Turtle DNA.

All sample results must remain distinct for future statistical analysis. The contractor must extract water filters using the Zymo Quick Miniprep Kit extraction kits (Zymo Research, Orange California) following a modification of the Spear et al. (2015) protocol. Next, they must amplify the DNA in a qPCR reaction using primers and thermocycler profile modified from Feist et al. (2018) to work with samples throughout the range of Alligator Snapping Turtles.

The contractor must visualize amplification using a QuantStudio3 machine (Thermo Fisher Scientific, Waltham MA). The samples must be run with a negative water control and internal positive control designed to capture the genetic variation present across the range of Alligator Snapping Turtles (interspecific and intraspecific) Vendors/laboratories must submit detailed capability statements that demonstrate how the same testing methods described above could be used.

Capability statements must also show how results would ensure consistency with prior testing in order to validate the overall research study findings. General capability statements should not be submitted. Vendors must demonstrate that they can perform the requested analysis following the requested methodology. Submit detailed capability statements by e-mail to Laniece Nixon at laniece.nixon@usda.gov with a courtesy copy to Keith Friot at keith.d.friot@usda.gov