535 Systematic Analysis of Existing and Novel Primers Toward Standardization of Skin Microbiome Research

Amplicon-based sequencing continues to be widely used for skin microbiome research. However, standard primers in use at most sequencing centers were originally optimized for the gut, despite the fact that the gut and skin microbiomes are extremely different in composition. In fact, standard primers, such as those spanning the V3V4 region, may not adequately capture Cutibacterium or Staphylococcus - two of the most prevalent genera on human skin. Continued use of these primers could contribute toward incomparable datasets and delay advances in our understanding of the skin microbiome. Here we combine a thorough in silico analysis of existing primers, followed by optimization and development of novel primers spanning the V1V3 and V3V4 regions. We then compare the performance of 6 primer pairs in a relevant complex mock community and in clinical samples obtained from human hands. As anticipated from in silico analyses, the commonly used V1V3 primer pair 27F/534R performed well in the mock community analysis, although underrepresenting S. capitis. The novel V1V3 primer 114F coupled with an optimized 519R primer (519Ropt) also performed well, picking up S. capitis better than 27F/534R, but underrepresenting N. flavescens and S. mitis; S. epidermidis was similarly overrepresented with both primer sets. Other primers tested, including new/optimized V3V4 primers, were less accurate. C. granulosum was underrepresented by an order of magnitude with all primer pairs tested. In clinical samples extracted from three human hands using the glove juice method, 27F/534R and 114F/519Ropt generally concurred with each other across the most abundant taxa, with 114F/519Ropt offering a possible benefit of additional Staphylococcus speciation. In conclusion, it is hoped this systematic analysis will advance skin microbiome research by enabling researchers to select appropriate primers to afford better cross-study comparisons in the future.