Diversity of Microbial Functional Genes Should Be Considered During the Interpretation of the qPCR Melting Curves

Soil microorganisms play an essential role in biogeochemical cycles. One approach to study these microbial communities is quantifying functional genes by quantitative PCR (qPCR), in which a melting curve analysis is usually assessed to confirm that a single PCR product is being quantified. However, the high diversity of functional genes in environmental samples could generate more than one peak in those curves, so the presence of two or multiple peaks does not always indicate nonspecific amplification. Here, we analyzed the taxonomic diversity of soil microorganisms harboring functional genes involved in nitrogen (N) and phosphorus (P) cycles, based on a database of genomes and metagenomes, and predicted the melting curve profiles of these genes. These functional genes were spread across many bacterial phyla, but mainly Proteobacteria and Actinobacteria. In general, the melting curves exhibited more than one peak or peaks with shoulders, mainly related to the variation of the nucleotide composition of the genes and the expected size of the amplicons. These results indicate that the melting curves of functional genes from environmental samples should be carefully evaluated, being in silico analyses a cost-effective way to identify inherent sequence diversity and avoid interpreting multiple peaks always as unspecific amplifications.