1. 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

2. 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

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

4. Workflow for metagenomic shotgun sequencing of skin microbiota

5. 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

6. 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