Improved single-swab sample preparation for recovering bacterial and phage DNA from human skin and wound microbiomes

Samuel Verbanic,Colin Y Kim,Irene A Chen,John M Deacon

doi:10.1186/s12866-019-1586-4

Abstract

BackgroundCharacterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples. While sample preparation from other microbiomes (e.g., gut) has been the subject of extensive optimization, procedures for skin and wound microbiomes have received relatively little attention. Here we describe an improved method for obtaining both phage and microbial DNA from a single skin or wound swab, characterize the yield of DNA in model samples, and demonstrate the utility of this approach with samples collected from a wound clinic.ResultsWe find a substantial improvement when processing wound samples in particular; while only one-quarter of wound samples processed by a traditional method yielded sufficient DNA for downstream analysis, all samples processed using the improved method yielded sufficient DNA. Moreover, for both skin and wound samples, community analysis and viral reads obtained through deep sequencing of clinical swab samples showed significant improvement with the use of the improved method.ConclusionUse of this method may increase the efficiency and data quality of microbiome studies from low-biomass samples.

Highlights

Characterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples
Recovery of phage and bacterial DNA released from swabs In this procedure, material from a single swab is separated into a Virus-like particle (VLP) fraction and ‘remainder’ fraction by centrifugation (Additional file 1: Figure S1)
The VLP fraction is treated with DNase I to digest free DNA, VLPs are precipitated, and capsids disrupted by sodium dodecyl sulfate (SDS) and proteinase K

Summary

Introduction

Characterization of the skin and wound microbiome is of high biomedical interest, but is hampered by the low biomass of typical samples. We describe an improved method for obtaining both phage and microbial DNA from a single skin or wound swab, characterize the yield of DNA in model samples, and demonstrate the utility of this approach with samples collected from a wound clinic. Methodological improvements in sample processing, sequencing, and bioinformatics are crucial to advance studies of the phylogenetic and functional diversity of the microbes colonizing the human body. The bacterial fraction of the microbiome is often the focus of such studies, revealing associations between bacterial community composition or gene expression and disease states [2]. Bioinformatic analysis has been a target for optimization, such as in comparing sequencing methods [10] and variable regions of the 16S rRNA gene to best analyze community composition [13, 14]

Methods

Results

Discussion

Conclusion