Strains were isolated using dilution-to-extinction cultivation in artificial lake medium supplemented with various nutrient and carbon sources. Positive well for Nanopelagicaceae were transferred and later grown in 400 - 500 ml culture medium until stationary phase. Cells were filtered onto 0.2 µm PES filters (Merck, Germany) and genomic DNA was purified using the Quick-DNA HMW MagBead Kit
(Zymo Research, USA). Paired-end sequencing (PE150) was performed on an Illumina Novaseq 6000 instrument by an external provider (Novogene, HK). Raw reads were quality trimmed using BBMap v36.x (
https://github.com/BioInfoTools/BBMap/) with default parameters and assembled with SPAdes v3.12.0 (using kmers 29, 49, 59, 69, 79, 89, 99, 109, 119, 127)31. Forty-eight genomes assembled in one circular contig, while the others had to be manually curated to circular chromosomes through repeated rounds of reads mapping to contigs using Geneious Prime 2022.1.1 (default mapper with high sensitivity;
www.geneious.com) and identifying overlapping ends.