Got Glycogen?: Development and Multispecies Validation of the Novel Preserve, Precipitate, Lyse, Precipitate, Purify (PPLPP) Workflow for Environmental DNA Extraction from Longmire's Preserved Water Samples.

Unfiltered and filtered water samples can be used to collect environmental DNA (eDNA). We developed the novel "Preserve, Precipitate, Lyse, Precipitate, Purify" (PPLPP) workflow to efficiently extract eDNA from Longmire's preserved unfiltered and filtered water samples (44-100% recovery). The PPLPP workflow includes initial glycogen-aided isopropanol precipitation, guanidium hypochlorite and Triton X-100-based lysis, terminal glycogen-aided polyethylene glycol precipitation, and inhibitor purification. Three novel eDNA assays that exclusively target species invasive to Australia were also developed: Tilapia_v2_16S concurrently targets (Mozambique tilapia) and (spotted tilapia) while R.marina_16S and C.caroliniana_matK discretely target (cane toad) and (fanwort), respectively. All 3 assays were validated before and validations using PPLPP workflow extracted samples. PPLPP workflow was concurrently validated and using all 3 assays. validations demonstrated that ) glycogen inclusion increased extracellular DNA recovery by ∼48-fold compared with glycogen exclusion, ) swinging-bucket centrifugation for 90 min at 3270 is equivalent to fixed-angle centrifugation for 5-20 min at 6750 , and ) Zymo OneStep Inhibitor Removal Kit, Qiagen DNeasy PowerClean Pro Cleanup Kit, and silica-Zymo double purification provide effective inhibitor removal. validation demonstrated 95.8 ± 2.8% (mean ± SEM) detectability across all 3 target species in Longmire's preserved unfiltered and filtered water samples extracted using the PPLPP workflow (without phenol:chloroform:isoamyl alcohol purification) after 39 d of incubation at room temperature and 50°C. PPLPP workflow is recommended for future temperate and tropical eDNA studies that use Longmire's to preserve unfiltered or filtered water samples.