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

Abstract

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 Oreochromis mossambicus (Mozambique tilapia) and Tilapia mariae (spotted tilapia) while R.marina_16S and C.caroliniana_matK discretely target Rhinella marina (cane toad) and Cabomba caroliniana (fanwort), respectively. All 3 assays were validated in silico before in vitro and in situ validations using PPLPP workflow extracted samples. PPLPP workflow was concurrently validated in vitro and in situ using all 3 assays. In vitro validations demonstrated that 1) glycogen inclusion increased extracellular DNA recovery by ∼48-fold compared with glycogen exclusion, 2) swinging-bucket centrifugation for 90 min at 3270 g is equivalent to fixed-angle centrifugation for 5-20 min at 6750 g, and 3) Zymo OneStep Inhibitor Removal Kit, Qiagen DNeasy PowerClean Pro Cleanup Kit, and silica-Zymo double purification provide effective inhibitor removal. In situ 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.

Keywords: eDNA; invasive species; method; tropical.

FIGURE 1.

Schematic overview of in silico, in vitro, and in situ validations conducted during PPLPP workflow development as well as PPLPP workflow overview (see Fig. 2 for detailed version of the PPLPP workflow). Arrows indicate the order in which in silico (light grey box), in vitro (medium grey boxes), and in situ (dark grey box) validations were conducted. In vitro validations 3–7 were used to derive the optimal PPLPP workflow (inset), which was then used for in vitro validations 8–11 and in situ validation (dashed lines). Target species and number of biologic replicates (n) are provided for each in vitro and in situ validation. Tilapia: O. mossambicus; cane toad: R. marina; Cabomba: C. caroliniana.

FIGURE 2.

Summary of the PPLPP workflow for unfiltered or filtered eDNA samples. Superscripts containing F_x, SF_x, or SP_x refer to corresponding Figure, Supplemental Figure, or Supplemental Protocol number, respectively. Optional PCI purification (step 13; grayed box) uses 1 phenol:chloroform:isoamylalcohol (25:24:1) and 2 chloroform:isoamylalcohol (24:1) phase separations. Hazardous substances are indicated by <!>. TE buffer, Tris-EDTA buffer. ^See Lysis Buffer (LB) I description (Supplemental Protocol 1 and Table 3). ^^See PEG8000-NaCl description (Supplemental Protocol 1 and Table 2). *For filters: vortex to dislodge material, transfer Longmire’s from both 2 ml LoBind tubes containing each filter half into one 50 ml LoBind tube, pulse spin to ensure complete transfer of Longmire’s, dilute combined volume up to 20 ml with DNA-free water, and then transfer both filter halves into one 2 ml LoBind tube (for step 9). **For filters: transfer 600 µl <!> LB < !> into 2 ml LoBind tube containing both filter halves (step 1c) to ensure complete lysis of filter-bound materials. ***For filters: transfer 600 µl <!> LB <!> into new 2 ml LoBind tube, pulse spin tube containing both filter halves, and transfer all residual <!> LB <!> into the same tube before terminal precipitation.

FIGURE 3.

PPLPP workflow in vitro validation of centrifugation, glycogen, and purification variables using R. marina and C. caroliniana low-copy clean-spike samples. Recovery efficiencies for R. marina gDNA (A–D) and C. caroliniana gDNA (E–H) were determined using R.marina_16S and C.caroliniana_matK assays under optimal conditions and species-specific gDNA standard curves for extrapolation (Table 4), respectively. The following PPLPP workflow variables were assessed: 1) initial precipitation centrifugation conducted in swinging-bucket rotor for 90 min (“SB_90 min”) or fixed-angle rotor for 5, 10, or 20 min (“FA_5 min,” “FA_10 min,” or “FA_20 min”; A, D, E, H); 2) low or high in-house glycogen concentration used in initial precipitation (44.4 µg/ml or 133.2 µg/ml; light or dark shading in A, B, E, F); and 3) exclusion or inclusion of terminal inhibitor purification using OneStep PCR Inhibitor Removal Kit (“Unpurified” or “Zymo”; solid or hashed bars in A and E), respectively. Different letter groupings in top and bottom letter rows (A and E) refer to significant differences (Tukey’s HSD P < 0.05) among and within centrifugation treatments, respectively, and different letters (B and F) refer to significant differences among treatments (Tukey’s HSD P < 0.05). Different letters (C and G) refer to a significant pairwise difference (2-tailed Welch’s t test P < 0.05). Treatment legends are provided (A, B, E, F). Bar plots represent mean ± SEM values.

FIGURE 4.

PPLPP workflow in vitro validation of glycogen and purification variables using R. marina and C. caroliniana low-copy tank-spike samples. Total O. mossambicus eDNA yield (nanograms ± SEM) is provided for multiple (A) and combined (A’) glycogen concentrations. Recovery of R. marina and C. caroliniana spiked-in gDNA (% ± SEM) is provided for multiple (B and C) and combined (B’ and C’) glycogen concentrations, respectively. Top and bottom letters indicate significant differences (Tukey’s HSD P < 0.05) among and within terminal inhibitor purification methods for each glycogen concentration tested (4.4, 22.2, and 44.4 µg/ml), respectively. Two-way ANOVAs for O. mossambicus (A), R. marina (B), and C. caroliniana (C) determined no glycogen effect within purification methods (bottom letters), whereas subsequent 1-way ANOVAs for O. mossambicus (A’), R. marina (B’), and C. caroliniana (C’) revealed significant species-specific differences among tested inhibitor purification methods. Unpurified: no terminal inhibitor purification; Zymo: OneStep PCR Inhibitor Removal Kit purification (Zymo Research Inc.); silica: silica purification (Supplemental Protocol 3); Qiagen: DNeasy PowerClean Pro Cleanup Kit purification (Qiagen Australia Pty Ltd).

FIGURE 5.

PPLPP workflow in situ validation of Longmire’s preservation effectiveness on multispecies eDNA collected in unfiltered (solid bars) and filtered (hashed bars) water samples from Ross River and subjected to 39 d of incubation at room temperature (RT; white bars) and 50°C (gray bars). Total eDNA yield (nanograms ± SEM; A) was determined for O. mossambicus, R. marina, and C. caroliniana in all treatments using Tilapia_v2_16S, R.marina_16S, and C.caroliniana_matK assays and species-specific gDNA standard curves (Table 4), respectively. Three-way ANOVA revealed no significant differences in eDNA yield among or within target species, capture methods, or incubation treatments (P > 0.3; see In situ validations). Detection rate (%; B) was subsequently determined for each target species (see Statistical analyses).