Amplicon sequencing with internal standards yields accurate picocyanobacteria cell abundances as validated with flow cytometry.

To understand ecosystem state and function, marine microbial ecologists seek measurements of organismal abundance and diversity at high taxonomic resolution. Conventional flow cytometry accurately estimates microbial cell abundance but only discerns broad groups with distinct optical properties. While amplicon sequencing resolves more comprehensive diversity within microbiomes, it typically only provides relative organismal abundances within samples, not absolute abundance changes. Internal genomic standards offer a solution for absolute amplicon-based measures. Here, we spiked genomic standards into plankton samples from surface seawater, gathered at 46-km intervals along a cruise transect spanning the southern California Current System and the oligotrophic North Pacific Subtropical Gyre. This enabled evaluation of the absolute volumetric gene copy abundances of 16S rRNA amplicon sequence variants (amplified with 515Y-926R universal primers, quantitatively validated with mock communities) and cell abundances of picocyanobacteria with known genomic 16S copy numbers. Comparison of amplicon-derived cell abundances of Prochlorococcus and Synechococcus with flow cytometry data from nearby locations yielded nearly identical results (slope = 1.01; Pearson's r = 0.9942). Our findings show that this amplicon sequencing protocol combined with genomic internal standards accurately measures absolute cell counts of marine picocyanobacteria in complex field samples. By extension, we expect this approach to reasonably estimate volumetric gene copies for other amplified taxa in these samples.