eDNA technology offers a non-invasive, replicable, and observer-independent method for monitoring biodiversity.
However, “eDNA” now refers to a wide range of protocols that vary greatly in sophistication – raising a critical question: are they all equally effective?
To explore this, we compared SPYGEN’s standard protocol for marine water environments (30 L, 12 PCR replicates, 1 million reads) with a simulated result of a basic protocol (2 L of water filtered, 8 PCR replicates, 100K reads) in a real marine site in Corsica.
- 40% of species went undetected and
- 100% of threatened species were missed with the basic protocol.
Why this matters?
Rigorous eDNA technologies are crucial. Inadequate methods may overlook rare species of high conservation or scientific value, resulting in misleading data and flawed conservation, management, or scientific conclusions.
When comparing eDNA protocols, keep in mind these 3 key elements:
- Sampling strategy matters: because eDNA is highly localized, filtering larger water volumes along integrated transects (vs. fixed points) captures more eDNA, providing a more complete and accurate biodiversity snapshot of the site.
- PCR replicates increase detection probability: using 12 PCR replicates means amplifying DNA in 12 independent subsamples, significantly boosting detection rates and minimizing false negatives, especially when paired with our high-performance primers.
- Sequencing depth drives detection power: The more DNA strands sequenced, the greater the chance of detecting species. Analyzing 1 million DNA reads greatly improves the detection of rare and low-abundance species, up to 10x more than standard 100,000-reads approaches.
Dive into the findings below:
Location: Corsica (France)
Ecosystem: Coastal, soft seabeds at seagrass meadows.
Depth: 40 m
Sampling method: 2.5 km transect, filtering 30 L of seawater using SPYGEN high-capacity filters.
Taxonomic group: fishes, via SPYGEN’s teleo primer.
Species and corresponding detected sequencing reads: see below.
| Common Name | Scientific Name | IUCN Status |
SPYGEN Protocol Liters of Water Filtered = 30L No of PCR Replicates = 12 Sequencing Depth = 1 million |
Basic Protocol Liters of Water Filtered = 2L No of PCR Replicates = 8 Sequencing Depth = 100 thousand |
|---|---|---|---|---|
| Longnose spurdog | Squalus blainville | DD | 11,733 | 1,173 |
| Round sardinella | Sardinella aurita | LC | 10,365 | 1,037 |
| Picarel | Spicara smaris | LC | 148,562 | 14,856 |
| Curlfin picarel | Spicara_flexuosa_S_smaris | LC | 30,044 | 3,004 |
| Bogue | Boops boops | LC | 158,364 | 15,836 |
| Annular seabream | Diplodus annularis | LC | 7,192 | 719 |
| White seabream | Diplodus sargus | LC | 8,176 | 818 |
| Axillary seabream | Pagellus acarne | LC | 5,188 | 519 |
| Common pandora | Pagellus erythrinus | LC | 5,121 | 512 |
| Red mullet | Mullus barbatus | LC | 3,304 | 330 |
| Comber | Serranus cabrilla | LC | 230 | Not Detected |
| Gilthead seabream | Sparus aurata | LC | 1,530 | Not Detected |
| Horse mackerel | Trachurus_mediterraneus_T_trachurus | LC/VU | 3,841 | 384 |
| Common dentex / Redband seabream / Red porgy | D. dentex_P. auriga_P. pagrus | VU/LC/LC | 12,677 | 1,268 |
| Starry ray / Thornback ray / Speckled ray | Raja_asterias_R_clavata_R_polystigma | LC/VU/LC | 657 | Not Detected |
| Marbled electric ray | Torpedo marmorata | VU | 652 | Not Detected |
| Marbled stingray | Dasyatis marmorata | NT | 23 | Not Detected |
| Common smooth-hound | Mustelus mustelus | EN | 748 | Not Detected |
| Common eagle ray | Myliobatis aquila | CR | 1,045 | Not Detected |
| Angelshark | Squatina squatina | CR | 124 | Not Detected |
This comparison is based on a simulated outcome derived from linear modelling of a simplified eDNA protocol and is intended for illustrative purposes only. It does not reflect the validated protocols of any specific provider. The aim is to demonstrate how key methodological parameters, such as water volume, number of PCR replicates, and sequencing depth, affect detection power and how easily species, including threatened ones, can be missed when protocols are underpowered.
The model evaluates expected species detection loss due to successive reductions in: (i) filtered water volume from 30 L to 2 L (15×), (ii) PCR replicates from 12 to 8 (1.5×), and (iii) sequencing depth from 1,000,000 to 100,000 reads (10×), all leading to reduction in usable eDNA signal. Estimated species loss is calculated based on the probability of falling below a detection threshold of 10 unique reads per species, used to avoid false positives. This is a theoretical exercise intended solely to illustrate general methodological considerations. It is not intended to evaluate, criticize, or compare the performance of any specific commercial provider.
Spygen’s standard protocol for marine and freshwater environments foresees filtering 30 liters of water per filter, with 2 filters used on the same site (i.e., 60 liters filtered in the same integrative transect), empirical experiences indicates that this approach further enhances detection power.
