CBIOMES researchers validating FCM‑anchored amplicon sequencing across ocean transects
Reporting by Helen Hill for CBIOMES
In their 2026 ISME Communications paper, Nathan Williams, Qicheng Bei, Yubin Raut, and Jed Fuhrman tackle a persistent limitation in marine microbial ecology: the fact that amplicon sequencing, despite its ubiquity, yields only relative abundances. For many ecological and biogeochemical questions, researchers need absolute counts—actual numbers of cells per volume of seawater—to understand population dynamics, quantify fluxes, and compare communities across space and time. Internal standards can provide these absolute values, but they are rarely used in large oceanographic surveys. Flow cytometry, by contrast, is routinely collected on major cruises and provides direct counts of key picocyanobacteria such as Synechococcus and Prochlorococcus. The authors ask whether these cytometric measurements can serve as an anchor to convert relative amplicon data into meaningful absolute abundances, effectively unlocking the vast archive of sequencing datasets that lack internal standards but do include flow cytometry.
To evaluate this idea, the team turned to the 29th Atlantic Meridional Transect (AMT29), a cruise that offered a rare opportunity: three independent ways to estimate microbial abundances were available simultaneously. Flow cytometry provided direct counts of Synechococcus and Prochlorococcus. Amplicon sequencing had been performed with internal standards, allowing absolute abundances to be reconstructed. And metagenomic data offered a third benchmark through single‑copy marker genes such as recA and radA. By anchoring the relative amplicon data to the flow‑cytometric counts of Synechococcus, the authors could test how well this simple scaling approach reproduced the two independent absolute‑abundance estimates.
The results were strikingly strong. The flow‑cytometry‑anchored amplicon estimates aligned closely with the internal‑standard‑corrected amplicon data, showing high correlations and slopes that indicated consistent scaling. They also matched the flow cytometry itself and agreed well with the metagenomic marker‑gene estimates. That this level of agreement emerged from anchoring to a single taxon—Synechococcus, chosen because its phycoerythrin signal is reliably detected in surface waters—suggests that flow cytometry can provide a robust reference point for reconstructing absolute abundances across diverse microbial groups.
Having validated the method, the authors applied it to the Global rRNA Universal Metabarcoding of Plankton (GRUMP) transects, which span the Pacific from the subarctic to the Southern Ocean and include extensive flow‑cytometry measurements. Using the same anchoring strategy, they generated absolute rRNA gene abundance estimates across these basin‑scale transects. Once again, the anchored estimates showed strong correspondence with independent flow‑cytometry counts, including for Prochlorococcus, even though the anchoring was based on Synechococcus. The correlations varied across transects but consistently demonstrated that the approach yields reasonable, ecologically interpretable absolute abundances.
The authors are careful to emphasize that these reconstructions are not exact cell counts. Instead, they represent well‑grounded estimates that capture the correct order of magnitude and spatial patterns. Yet this level of accuracy is more than sufficient for many ecological and modeling applications. The implications are substantial. Absolute microbial abundances are essential for constraining ecosystem models, interpreting biogeochemical fluxes, and understanding how microbial communities structure the ocean’s metabolic landscape. Because flow cytometry is widely collected on cruises such as AMT, HOT, BATS, GEOTRACES, and many others, this method provides a practical way to convert enormous quantities of existing relative‑abundance data into the absolute terms that models require.
Publication:
Nathan L R Williams, Qicheng Bei, Yubin Raut, Jed A Fuhrman (2026), Converting Relative Amplicon Abundances to Absolute Abundances via Flow Cytometry: Metagenomic Validation and Application to Long Ocean Transects, ISME Communications, https://doi.org/10.1093/ismeco/ycag081