Evaluating cpn60 for high-resolution profiling of the mammalian skin microbiome and detection of phylosymbiosis

Abstract

Abstract Despite being the most widely used phylogenetic marker for amplicon-based profiling of microbial communities, limited phylogenetic resolution of the 16S rRNA gene limits its use for studies of host-microbe co-evolution. In contrast, the cpn60 gene is a universal phylogenetic marker with greater sequence variation capable of species-level resolution. This research compared mammalian skin microbial profiles generated from cpn60 and 16S rRNA gene sequencing approaches, testing for patterns of phylosymbiosis that suggest co-evolutionary host-microbe associations. An ~560 bp fragment of the cpn60 gene was amplified with universal primers and subjected to high-throughput sequencing. Taxonomic classification of cpn60 sequences was completed using a naïve-Bayesian QIIME2 classifier created for this project, trained with an NCBI-supplemented curated cpn60 database (cpnDB_nr). The cpn60 dataset was then compared to published 16S rRNA gene amplicon data. Beta diversity comparisons of microbial community profiles generated with cpn60 and 16S rRNA gene amplicons were not significantly different, based on Procrustes analysis of Bray-Curtis and UniFrac distances. Despite similar relationships among skin microbial profiles, improved phylogenetic resolution provided by the cpn60 gene sequencing permitted observations of phylosymbiosis between microbial community profiles and their mammalian hosts that were not previously observed with 16S rRNA gene profiles. Subsequent investigation of Staphylococcaceae taxa using the cpn60 gene showed increased phylogenetic resolution compared the 16S rRNA gene profiles, revealing potential co-evolutionary host-microbe associations. Overall, our results demonstrate that 16S rRNA and cpn60 marker genes generate comparable microbial community composition patterns while cpn60 better facilitates analyses, such as phylosymbiosis, that require increased phylogenetic resolution.