Evaluation of Methods for the Extraction of Microbial DNA From Vaginal Swabs Used for Microbiome Studies

• Published in: Frontiers in cellular and infection microbiology Vol. 9; p. 197
• Main Authors: Mattei, Valentina, Murugesan, Selvasankar, Al Hashmi, Muna, Mathew, Rebecca, James, Nicola, Singh, Parul, Kumar, Manoj, Lakshmanan, Arun Prasath, Terranegra, Annalisa, Al Khodor, Souhaila, Tomei, Sara
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 06.06.2019
• Subjects: 16S sequencing; Adult; Biodiversity; Cellular and Infection Microbiology; DNA extraction; DNA, Bacterial - isolation & purification; DNA, Ribosomal - genetics; Female; High-Throughput Nucleotide Sequencing; Humans; metagenomics; Metagenomics - methods; microbiota; Microbiota - genetics; Real-Time Polymerase Chain Reaction; RNA, Ribosomal, 16S - genetics; Vagina - microbiology; vaginal swabs; microbiota; DNA extraction; vaginal swabs; metagenomics; 16S sequencing
• ISSN: 2235-2988; 2235-2988
• DOI: 10.3389/fcimb.2019.00197

The composition of the microbiome in human body sites plays an important role in health. The vaginal environment is colonized by several species of bacteria that have a major influence on reproductive health. The advancement of sequencing technologies has made the assessment of the composition of the microbiota possible through microbial DNA extraction and sequencing. Therefore, it is of a paramount importance to select a sensitive and reproducible DNA extraction method, that facilitates isolation of microbial DNA with a sufficient quantity and purity, from microbial species living in the vaginal environment. Here, we have evaluated four different DNA extraction protocols from self-collected vaginal swabs. Five healthy female volunteers were enrolled in the study. Each donor was asked to self-collect 4 samples using Copan ESwab. DNA was extracted using Qiagen DNeasy kit and three modified protocols of the MoBio PowerSoil kit ("DNeasy PowerSoil" after acquisition from Qiagen). DNA quantity and integrity was checked through Nanodrop and LabChip GX. DNA was further tested through quantitative real-time PCR (qPCR) and 16S sequencing. Vaginal microbiota diversities were determined using MiSeq-Illumina high-throughput sequencing of bacterial 16S rDNA V1-V3 fingerprint. Sequencing data were analyzed using QIIME pipeline. Qiagen DNeasy protocol resulted in the highest DNA yield as compared to the modified protocols of MoBio Powersoil kit. The size of the DNA extracted using each protocol was ~40 kb. Qiagen DNeasy protocol gave the highest Genomic Quality Score (average ± standard deviation: 4.24 ± 0.36), followed by the different MoBio Powersoil protocols. A substantial variability in microbial DNA abundance was found across the protocols. The vaginal microbiota of the healthy volunteers was dominated by . MoBio Powersoil kit provided a significantly higher alpha diversity as compared to the Qiagen DNeasy kit, while beta diversity measures did not reveal any significant cluster changes, except when the Bray-Curtis method was applied. We were able to isolate microbial DNA from the vaginal swabs. Qiagen DNeasy method gave the highest DNA yield and quality but was not optimal in detecting microbial diversity. The modified MoBio PowerSoil protocols showed higher microbial diversities as compared to the standard protocol.