Research Techniques Made Simple: Profiling the Skin Microbiota Max D. Grogan, Casey Bartow-McKenney, Laurice Flowers, Simon A. B. Knight, Aayushi Uberoi, Elizabeth A. Grice# Departments of Dermatology & Microbiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, USA #Corresponding author

Collection and processing of specimens for culture-dependent and culture-independent studies Multiple specimens are collected using the same method of choice (pictured is a swab) Negative controls are included to assess background and reagent contamination throughout the process Specimens for culture-dependent analysis are streaked on a plate containing growth media Specimens for culture-independent analysis are subjected to bulk genomic DNA extraction

Workflow for amplicon-based sequencing (eg Workflow for amplicon-based sequencing (eg. bacterial 16S rRNA gene sequencing)

Workflow for metagenomic shotgun sequencing of skin microbiota

Advantages of culture-independent approaches to profile skin microbiota • Does not require growth and isolation of microorganisms in culture • High-throughput and quantitative approach for community-wide analysis of skin microbiota • Powerful hypothesis-generating tool • Bioinformatic tools and reference datasets are readily available to analyze and visualize microbiome datasets • Shotgun metagenomic sequencing approaches provide enhanced taxonomic resolution and functional insights based on genetic and metabolic pathway reconstructions. • Identification of new mutations in individual cells

Limitations of culture-independent approaches to profile the skin microbiota • Cannot distinguish live versus dead microorganisms • Skin specimens are especially prone to contamination and false positives • Sequencing data are associative and cannot establish causality • Reference datasets are limited for skin microorganisms