The normalization of gene abundance affects the discovery: A case of metal resistance genes in coastal sediments

The advent of metagenomic technologies has given rise to a plethora of gene abundance calculation methods, which have emerged in a successive manner. Nevertheless, the extent to which these approaches are appropriate for metagenomic data and the specific research objectives remain subjects of debate. In this study, metagenomics sequencing was utilised to analyse the sediments from two coastal regions of China: the Beibu Gulf (BBG) and the Liaohe Estuary (LHE). The abundance of metal resistance genes (MRGs), which are critical genetic elements that influence ecosystem functioning and health, in the regions under study was obtained by two different calculation methods: the cell-normalized abundance (copies per prokaryotic cell) and the ppm abundance (sequences per million sequences). The application of diverse abundance calculation methodologies yielded markedly divergent outcomes. The cell-normalized and ppm abundances indicated a substantial elevation of total MRG abundance in the BBG and LHE sediments, respectively. Furthermore, disparate comparison outcomes were identified based on the varied abundance calculation methodologies employed across distinct MRG types, including genes demonstrating resistance to Fe, Al, Cr, Se, and multi-metals. These disparities can be ascribed to the variations in the prokaryotic biomass among different sediments, signifying that it is imperative to select an appropriate abundance calculation method according to whether the research objective necessitates the consideration of biomass differences. Furthermore, the results of multiple correlation analyses indicated that petroleum content was the key factor, and the ruvB gene was identified as an indicator for the MRG level based on both two abundance calculation methods. These findings establish a methodological foundation for metagenomics-based research and offer insights into the MRGs present in coastal sediments.