Abstract
The human skin is home to a complex community of microorganisms that include eukaryotes, bacteria, and fungi that collectively make up the skin microbiome. Despite years of research, little is known about these different species of microbes that colonize the skin’s surface or within sebaceous glands and their roles in inflammatory skin disorders. Advances in 16s and whole metagenomic shotgun (WMS) sequencing technologies have provided researchers with the tools to study microbial taxonomy and differences in phylogroups on the skin like never before. However, the quality of sequencing results is based on efficient extraction of genomic DNA. DNA extraction is an important first step prior to sequencing and analysis in identifying and characterizing microbes on the skin. DNA extraction is a multi-step process: (1) Sample collection and storage, (2) Lysis of bacterial cell membranes or walls, (3) Use of DNA extraction kits to isolate genomic DNA. Currently, DNA extractions in skin-microbiome research are inconsistent regarding the use of lysis methods and kit choice. Enzymatic, mechanical, heat, and freezing lysis conditions may have considerable effects on DNA quality and yield, so finding these optimal conditions are crucial for experimental design and downstream sequencing results. Because these methods are not standardized, lysis efficiency may vary from method to method, and could potentially introduce bias across samples that were treated differently. Furthermore, because the skin is predominantly colonized by Gram-positive bacteria that have cell walls composed of thick layers of peptidoglycan and are harder to lyse, we suspect these species are inaccurately represented. We hypothesize that modifications to DNA extraction kits differentially affect yield of bacterial genomic DNA being sampled.
We investigated our hypothesis by the following objectives:I. Characterize the effects of different variations to DNA extraction methods on cell viability, quantity, and quality of yield for Staphylococcus epidermidis (n = 3 for all bacteria in each condition tested).
A. Viability from mid-logarithmic phase cultures by CFU using variations of: (i) freeze-thaw, (ii) heat lysis, (iii) lysozyme concentration, incubation time, and temperature.
B. DNA quantity by Qubit from mid-log phase cultures using variations of: (i) freeze-thaw, (ii) heat lysis, (iii) lysozyme concentration, incubation time, and temperature.
II. Determine the extraction efficiencies between various DNA extraction methods for Staphylococcus epidermidis and Cutibacterium acnes (n = 5 for all bacteria in each method tested).
A. DNA quantity by Qubit from mid-logarithmic phase cultures using the following DNA extractions methods: (i) Enzsteel, (ii) ReadyLyse Steel, and (iii) a method consisting of the conditions of freeze-thaw, heat lysis, lysozyme concentration, and incubation time from objective I that yielded the best results.