Filter pore size influences taxonomic composition of retained eDNA from seawater samples—evidence from shotgun sequencing

Filter pore size was associated with the taxonomic composition of environmental DNA retained from seawater samples in a comparative study that paired shotgun sequencing with 18S ribosomal DNA metabarcoding. In samples collected at Skovshoved Harbour in Copenhagen in October 2017, filters with 5.0- and 8.0-micrometre pores retained a higher proportion of eukaryotic reads and a lower proportion of bacterial and archaeal reads than filters with 0.2- and 1.2-micrometre pores.

Researchers collected 15 one-litre samples of surface seawater, with three biological replicates for each of five filter configurations: open polyethersulfone filters with pore sizes of 0.2, 1.2, 5.0 and 8.0 micrometres and enclosed 0.2-micrometre Sterivex filters. The open filters had a filtration area of about 17 square centimetres, compared with about 10 square centimetres for the enclosed filters. Pore sizes were selected in relation to expected eukaryotic cell sizes of 10–100 micrometres and bacterial sizes of 0.1–5.0 micrometres. The study tested whether larger pores would increase the proportion of eukaryotic eDNA.

Each sample was subjected to parallel shotgun sequencing and targeted 18S metabarcoding. Shotgun libraries were sequenced on an Illumina NovaSeq 6000, while the metabarcoding workflow amplified an approximately 110-base-pair region of the 18S gene. DNA was extracted with a modified Qiagen Blood & Tissue kit protocol. The shotgun analysis produced 394.4 million raw reads, of which 96.7% were merged, yielding 190.6 million paired reads. After database matching and filtering for significance, sequence similarity, query coverage and alignment length, 9.0 million reads, or 2.3% of the paired reads, remained for further analysis.

At the superkingdom level, taxonomic composition differed between small and large pores. Filters with 0.2- and 1.2-micrometre pores had mean bacterial and eukaryotic read proportions of 63% and 28%, respectively, whereas the corresponding proportions for 5.0- and 8.0-micrometre filters were 31% and 49%. Wilcoxon tests with Benjamini–Hochberg-adjusted p-values indicated more archaeal and bacterial reads with the small pores and more eukaryotic and viral reads with the large pores. An ordination based on the presence or absence of phyla also separated the samples by pore size.

Classification of the eukaryotic subset was more limited. Among eukaryotic reads, 16.4% were assigned to a kingdom, equivalent to 5.7% of the reads searched against BLAST and 0.8% of all paired shotgun reads. Small-pore filters produced more reads assigned to Viridiplantae, while large-pore filters produced more reads assigned to Metazoa, Fungi and other eukaryotic groups. The investigators did not directly measure which forms of eukaryotic eDNA were preferentially retained or the passage of bacterial cells and cellular debris through the filters.

Shotgun sequencing identified 93 phyla in total across Archaea, Bacteria, Eukaryota and Viruses, while the targeted metabarcoding analysis detected 45 eukaryotic phyla. The eukaryotic comparison showed substantial overlap: 39 phyla were detected by both methods, six were unique to metabarcoding and nine were unique to shotgun sequencing. Shotgun sequencing therefore recovered 48 eukaryotic phyla in total, compared with 45 in metabarcoding. The method-specific detections may reflect differences in amplification, reference-database coverage and unrecorded sequence diversity, so the comparison does not establish that either method consistently detects every biological phylum present.

The researchers also compared shotgun reads assigned to genera in four marine metazoan groups with genera recorded from Denmark. Among shotgun detections, 12 of 13 polychaete genera, six of seven malacostracan genera, 11 of 18 bivalve genera and 23 of 59 actinopterygian genera were classified as Danish. Corresponding metabarcoding results were 22 of 23, three of four, six of six and five of six. The authors regarded exotic-genus assignments as more likely incorrect identifications caused by incomplete reference coverage than as genuine Danish records. Reads assigned to exotic genera were consistently less abundant than those assigned to Danish genera, particularly in large-pore samples, but the study did not establish a universal read-count threshold for separating the categories.

Important limitations constrain the findings. The experiment covered one harbour and one sampling period, with only three biological replicates per filter configuration and no negative field control. The third replicate differed from the first two across filter types, including higher bacterial abundance in several phyla and lower abundance in Discosea and Echinodermata. This pattern was consistent with short-term temporal or spatial variation in coastal eDNA, but the design could not test that explanation formally. Contamination or background DNA also could not be ruled out.

Together, the results support an association between pore size and retained taxonomic composition in the seawater samples examined. They also show that shotgun sequencing can recover broad eukaryotic diversity at the phylum level despite a low overall assignment rate. The findings do not establish a universal optimum filter pore size for eDNA sampling, and the low assignment rate, limited replication, single location and absence of field controls remain central limitations of the study.

Subject of Research: Effects of filter pore size on the taxonomic composition of shotgun-sequenced seawater environmental DNA

Article Title: Filter pore size influences taxonomic composition of retained eDNA from seawater samples—evidence from shotgun sequencing

Article References: Gómez-Repollés, A., Sigsgaard, E. E., Jensen, M. R., & Thomsen, P. F. (2026). Filter pore size influences taxonomic composition of retained eDNA from seawater samples—evidence from shotgun sequencing. Metabarcoding and Metagenomics, 10, Article e164232. https://doi.org/10.3897/mbmg.10.164232

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.164232

Keywords: environmental DNA, seawater, filter pore size, shotgun sequencing, 18S metabarcoding, eukaryotes, bacteria, taxonomic composition, biomonitoring, Filter, pore, size