Each Cys-containing protein sequence in the NR database was initially searched against the Olavius symbionts’ metagenomic database for possible TGA/TAG/TAA-containing homologs using TBLASTN with default parameters. Only local alignments, in which Cys in the query protein was aligned with TGA codon in the nucleotide sequence from the Olavius symbionts’ metagenomic database, were selected for further analysis. For each TGA-containing nucleotide sequence identified in the metagenomic database, regions upstream and downstream of the putative in-frame TGA codon were analyzed to identify a minimal ORF. If a stop codon was found between the in-frame TGA codon and an initiation codon (ATG or GTG), such a TGA-containing sequence was discarded.