Intent to Sole Source-Alligator Snapping Turtle eDNA Analysis from Filtered Water Samples

The USDA Forest Service intends to issue a firm fixed, price, sole source Blanket Purchase Agreement (BPA) for analyses of environmental DNA samples for alligator snapping turtles. 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 to a laboratory in an insulated container containing cold packs for analysis. Each tube should be considered as an independent sample. The laboratory is required to detect sequences of Alligator Snapping Turtle DNA from filter medium and ensure no cross-contamination of samples.

The laboratory must provide a detailed report showing CT scores for each run (3 runs per sample) and whether 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 a thermocycler profile modified by 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 negative water control and internal positive control designed to capture the genetic variation present across the range of Alligator Snapping Turtles (interspecific and intraspecific). Establishing the BPA on a sole source basis is warranted in this situation, as Tangled Bank Conservation is the only vendor with the necessary testing primers and protocols in place. These are necessary to ensure DNA analyses and Quantitative PCR services meet all research study requirements for alligator snapping turtle genetic analysis.