Thalassiosira gravida single-cell microbiomes

Primary production in aquatic systems depends on the interactions between microalgae and their associated bacteria. Current knowledge of algal microbiomes largely comes from studies on natural mixed communities or axenic algal monocultures, approaches that overlook the influence of genotypic diversity within algal hosts and its role in shaping associated bacterial communities. To address this gap, we established a single-cell 16S rRNA sequencing workflow combined with CRISPR-Cas9 guided depletion of chloroplast-derived 16S sequences. We validated this method by analyzing the bacterial communities of 144 single cells from three genotypes of the Arctic diatom Thalassiosira gravida cultivated under varying environmental conditions. Of these, 62 single cells were further subjected to CRISPR-Cas9 treatment, which enhanced sequencing depth and increased estimates of bacterial richness per individual diatom cell by up to 56%. This improved resolution uncovered not only genotype-specific associations between host cells and their microbiota but also rare bacterial taxa that remained undetected in conventional 16S metabarcoding.