Over the past two decades, environmental DNA (eDNA) analysis has become a crucial tool for monitoring aquatic ecosystems. The most common method, metabarcoding, relies on PCR amplification of a smaller genetic region to identify specific taxa. However, PCR can lead to "significant taxonomic bias" because it often amplifies the DNA of different organisms unequally, making quantitative estimates difficult.
To avoid this, scientists have increasingly explored "shotgun sequencing"- a broad approach that sequences the DNA in a sample much more broadly - across the entire tree of life and across the genome. Unfortunately, in marine environments, shotgun sequencing is typically overwhelmed by microbial DNA, burying the genetic traces of less abundant macro-organisms such as animals.
Bigger Pores, Better Animal DNA Capture?
In a new study published in Metabarcoding and Metagenomics , researchers investigated if they could capture a higher proportion of eukaryotic (animal and plant) DNA simply by using filters with larger pore sizes.
Filter pore sizes are expected to influence results, since eDNA may be present in many different states, including but not limited to complete organisms, sloughed tissue, feces, free DNA, or gametes.
- commented Dr. Adrián Gómez-Repollés, the lead author of the study
To test this, the team collected 15 seawater samples from Skovshoved Harbour in Denmark and filtered them using pore sizes ranging from 0.2 µm to 8.0 µm. The results showed a stark contrast in the type of DNA captured based on the filter size:
Filters with smaller pore sizes (0.2 µm and 1.2 µm) retained a significantly greater proportion of bacterial reads than eukaryotic reads (63% vs. 28%); conversely, filters with larger pore sizes (5.0 µm and 8.0 µm) retained a significantly greater proportion of eukaryotic reads than bacterial reads (49% vs. 31%).
By switching to 5.0 µm or 8.0 µm filters, the researchers successfully reduced the dominance of bacteria. Of the 19 metazoan (animal) phyla detected using shotgun sequencing, all but one were found to be more abundant when using the larger pore sizes.
Looking to the Future of Biomonitoring
When compared alongside traditional 18S rDNA metabarcoding, the shotgun sequencing method successfully shared 39 of the 54 detected eukaryotic phyla, indicating a similar performance in detecting the presence of high-level taxonomic groups.
To test the potential of shotgun sequencing in applied biomonitoring even further, the researchers examined the results at genus-level for a number of well known marine animals such as fish, mussels, crustaceans and bristle worms. Here, they found both DNA matches to native Danish species but also to exotic taxa that were highly unlikely and probably due to the low level of resolution in shotgun sequencing, where genetic regions of low variation and coverage are sequenced. But, when they looked further into the results they observed that the local taxa consistently comprised a higher number of reads. The number of reads could thus be a simple way to initially separate authentic taxa from erroneous matches in eDNA studies based on shotgun sequencing, although the approach needs further testing.
However, the authors note current limitations with the technology, primarily driven by incomplete public DNA reference databases. In the study, only 0.78% of the total shotgun reads could be definitively assigned to a superkingdom level. Another drawback was the lack of field controls to rule out cross-contamination or input from airborne DNA, coupled with a limited spatiotemporal design involving only a single sampling location.
Despite these hurdles, shotgun sequencing and filters with larger pore sizes could potentially be a significant step forward for eDNA in marine biology. As global genomic databases continue to expand, "the taxonomic coverage and resolution of shotgun sequencing should improve, likely enhancing the potential of shotgun sequencing for future eDNA research", says Philip Francis Thomsen, professor and senior author on the study.
Original study:
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. https://doi.org/10.3897/mbmg.10.164232
