Developing a microfluidic-based epicPCR reveals diverse potential hosts of the gene in marine cold seep.

Anaerobic methanotrophic (ANME) microbes play a crucial role in the bioprocess of anaerobic oxidation of methane (AOM). However, due to their unculturable status, their diversity is poorly understood. In this study, we established a microfluidics-based epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) to fuse the 16S rRNA gene and gene to reveal the diversity of ANME microbes ( gene hosts) in three sampling push-cores from the marine cold seep. A total of 3725 16S amplicon sequence variants (ASVs) of the gene hosts were detected, and classified into 78 genera across 23 phyla. Across all samples, the dominant phyla with high relative abundance (>10%) were the well-known , and some bacterial phyla such as , , and ; however, the specificity of these associations was not verified. In addition, the compositions of the gene hosts were significantly different in different layers, where the archaeal hosts increased with the depths of sediments, indicating the carriers of AOM were divergent in depth. Furthermore, the consensus phylogenetic trees of the gene and the 16S rRNA gene showed congruence in archaea not in bacteria, suggesting the horizontal transfer of the gene may occur among host members. Finally, some bacterial metagenomes were found to contain the gene as well as other genes that encode enzymes in the AOM pathway, which prospectively propose the existence of ANME bacteria. This study describes improvements for a potential method for studying the diversity of uncultured functional microbes and broadens our understanding of the diversity of ANMEs.