In recent years, environmental DNA (eDNA) analysis has emerged as a revolutionary approach for monitoring aquatic ecosystems, by allowing the detection of organisms through genetic material shed into the environment. Among the various techniques, metabarcoding has been widely adopted due to its ability to amplify and sequence specific genetic regions, thus identifying a spectrum of taxa present in water samples. However, despite its popularity, metabarcoding suffers from inherent biases linked to PCR amplification, which can skew the taxonomic composition and impede accurate quantitative estimates of biodiversity.
PCR amplification bias, a well-known limitation in metabarcoding, arises because PCR primers tend to favor the amplification of some taxa over others, leading to unequal representation in the sequencing data. This issue complicates efforts to obtain a true ecological snapshot, especially when quantitative assessments of taxa abundance are crucial for ecosystem monitoring. To circumvent these issues, researchers have increasingly turned toward shotgun sequencing, a method that sequences all DNA fragments in a sample without bias toward any specific genetic region.
Shotgun sequencing, by capturing a broader array of genetic information, theoretically allows for more accurate and less biased assessments of biodiversity in environmental samples. Yet, the application of this approach in marine ecosystems faces a major hurdle: bacterial DNA overwhelmingly dominates seawater DNA samples, posing a significant challenge to detecting less abundant macro-organisms like fish, crustaceans, or marine plants. Effectively separating eukaryotic DNA from the microbial background remains one of the biggest obstacles limiting shotgun sequencing’s potential in marine biomonitoring.
A recent groundbreaking study published in Metabarcoding and Metagenomics by Dr. Adrián Gómez-Repollés and colleagues has taken significant strides toward overcoming this hurdle through a simple yet ingenious approach—manipulating filter pore sizes used in eDNA sampling. The researchers hypothesized that filter pore size would substantially influence the taxonomic composition of DNA retained from seawater samples by preferentially capturing either smaller microbial DNA fragments or larger pieces of eukaryotic DNA associated with organisms or biological material.
To rigorously test this hypothesis, the team collected fifteen seawater samples from Skovshoved Harbour, Denmark. They filtered the water through a series of membrane filters featuring a gradient of pore sizes, ranging from ultra-fine 0.2 micrometers up to relatively large 8.0 micrometers. This range was designed to capture DNA in different physical states—from free-floating DNA fragments and microbial cells to larger eukaryotic particles such as sloughed tissue, fecal matter, or whole small organisms. By carefully comparing the taxonomic profiles yielded by each pore size, the researchers sought to uncover how filter choice shapes the overall recovery of biodiversity signals.
The results revealed a striking pattern. Filters with smaller pore sizes, specifically 0.2 µm and 1.2 µm, predominantly trapped bacterial DNA, which constituted about 63% of retained genetic material. Eukaryotic DNA, including that from animals and plants, accounted for only 28% under these conditions. Conversely, filters with larger pores of 5.0 µm and 8.0 µm substantially shifted this balance, capturing a greater proportion of eukaryotic DNA—49%—while bacterial reads dropped to 31%. This pivotal observation demonstrated that increasing filter pore sizes could effectively reduce bacterial DNA ‘noise’ and enrich samples for macro-organism DNA.
Looking deeper, the team’s shotgun sequencing analysis uncovered a richer representation of animal biodiversity when using larger pore size filters. Among 19 animal phyla detected, every phylum except one was more abundant in samples processed through the 5.0 µm and 8.0 µm filters. This finding is revolutionary because it charts a clear pathway to improving shotgun sequencing protocols for marine biomonitoring, shifting the method closer to practical, high-resolution applications in ecological surveys and conservation efforts.
To benchmark shotgun sequencing against the conventional metabarcoding approach, the study performed a comparative taxonomic inventory. Both methods detected a substantial, overlapping set of 39 eukaryotic phyla out of 54 identified, confirming shotgun sequencing’s robustness for broad taxonomic assessments. However, the shotgun approach also uncovered eukaryotic groups not detected by metabarcoding, highlighting the complementary nature of these techniques. This interplay suggests that shotgun sequencing, despite current limitations, holds great potential for expanding the taxonomic breadth accessible in eDNA studies.
At the genus-level resolution, the researchers zoomed in on iconic marine taxa including fish, mussels, crustaceans, and polychaete worms native to Danish waters. Shotgun sequencing identified DNA sequences that corresponded to expected local species, reinforcing the method’s ecological validity. Simultaneously, however, some unexpected or “exotic” taxa also appeared, likely artifacts linked to shotgun sequencing’s lower resolution and sequencing of less variable genomic regions. Importantly, the relative read abundances were skewed: native species consistently generated higher sequencing depths compared to spurious matches, suggesting that read count thresholds might aid in distinguishing true positive identifications from noise.
Despite the encouraging findings, the authors acknowledge key challenges currently inhibiting the widespread adoption of shotgun sequencing for marine biomonitoring. Chief among these is the incomplete DNA reference underpinning public databases, which limits the confident assignment of sequences. In this study, less than 1% of shotgun reads could be definitively classified at the superkingdom taxonomic level. This technical bottleneck underscores the critical need for continued expansion and refinement of reference genomes, particularly from marine eukaryotes. Additionally, the study lacked field controls to exclude contamination or airborne DNA inputs, and its spatial sampling was restricted to a single location, limiting conclusions about broader ecological patterns.
Nonetheless, the findings paint an optimistic picture. Increasing filter pore sizes in eDNA collection could represent an easily implementable strategy to enrich shotgun sequencing libraries for diverse macro-organisms in marine environments, addressing the major hurdle of bacterial dominance. Moreover, as global genomic resources mature and analytical pipelines refine, shotgun sequencing’s taxonomic resolution and coverage will continue to improve, unlocking new frontiers in environmental surveillance.
Philip Francis Thomsen, the senior author of the study, emphasizes this future potential: “The expansion of genomic databases coupled with refined methodological workflows positions shotgun sequencing to transform marine eDNA research, enhancing biodiversity assessments while reducing biases inherent in PCR-based approaches.” He envisions a time when near-complete taxonomic inventories can be rapidly generated from seawater samples, empowering conservationists and resource managers in their efforts to protect fragile marine ecosystems from multiple anthropogenic threats.
In conclusion, this pioneering research demonstrates that subtle methodological choices—in this case, filter pore size—can profoundly impact the quantity and quality of biodiversity information extracted through shotgun sequencing of seawater eDNA. By strategically selecting larger pore filters, scientists can mitigate bacterial interference and enhance the detection of ecologically and economically critical marine taxa. This work heralds a new era of environmental DNA monitoring, where unbiased, broad-scale genetic surveillance becomes a cornerstone of marine ecosystem stewardship and biodiversity conservation worldwide.
Subject of Research: The influence of filter pore size on the taxonomic composition of environmental DNA retained from seawater samples using shotgun sequencing methods.
Article Title: Filter pore size influences taxonomic composition of retained eDNA from seawater samples—evidence from shotgun sequencing
News Publication Date: February 18, 2026
Web References:
DOI: 10.3897/mbmg.10.164232
Journal: Metabarcoding and Metagenomics
References:
Gómez-Repollés A, Sigsgaard EE, Jensen MR, Thomsen PF (2026) Filter pore size influences taxonomic composition of retained eDNA from seawater samples—evidence from shotgun sequencing. Metabarcoding and Metagenomics 10: e164232.
Image Credits: Gómez-Repollés et al., 2026
Keywords: environmental DNA, eDNA, shotgun sequencing, metabarcoding, filter pore size, marine biomonitoring, taxonomic bias, PCR limitations, biodiversity assessment, marine ecosystems, eukaryotic DNA, bacterial DNA dominance
