Evaluation of methods for the extraction of microbial DNA from vaginal swabs used for microbiome studies

Valentina Mattei, Selvasankar Murugesan, Muna Al Hashmi, Rebecca Mathew, Nicola James, Parul Singh, Manoj Kumar, Arun Prasath Lakshmanan, Annalisa Terranegra, Souhaila M. Al Khodor, Sara Tomei

Research output: Contribution to journalArticle

Abstract

Background: 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. Methods: 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. Results: 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 Lactobacillus species. 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. Conclusion: 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.

Original languageEnglish
Article number197
JournalFrontiers in Cellular and Infection Microbiology
Volume9
Issue numberJUN
DOIs
Publication statusPublished - 1 Jan 2019

Fingerprint

Microbiota
DNA
Healthy Volunteers
Reproductive Health
Dermatoglyphics
Lactobacillus
Ribosomal DNA
DNA Sequence Analysis
Human Body
Real-Time Polymerase Chain Reaction
Tissue Donors
Technology
Bacteria

Keywords

  • 16S sequencing
  • DNA extraction
  • Metagenomics
  • Microbiota
  • Vaginal swabs

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Microbiology (medical)
  • Infectious Diseases

Cite this

Evaluation of methods for the extraction of microbial DNA from vaginal swabs used for microbiome studies. / Mattei, Valentina; Murugesan, Selvasankar; Al Hashmi, Muna; Mathew, Rebecca; James, Nicola; Singh, Parul; Kumar, Manoj; Prasath Lakshmanan, Arun; Terranegra, Annalisa; Al Khodor, Souhaila M.; Tomei, Sara.

In: Frontiers in Cellular and Infection Microbiology, Vol. 9, No. JUN, 197, 01.01.2019.

Research output: Contribution to journalArticle

Mattei, Valentina ; Murugesan, Selvasankar ; Al Hashmi, Muna ; Mathew, Rebecca ; James, Nicola ; Singh, Parul ; Kumar, Manoj ; Prasath Lakshmanan, Arun ; Terranegra, Annalisa ; Al Khodor, Souhaila M. ; Tomei, Sara. / Evaluation of methods for the extraction of microbial DNA from vaginal swabs used for microbiome studies. In: Frontiers in Cellular and Infection Microbiology. 2019 ; Vol. 9, No. JUN.
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abstract = "Background: 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. Methods: 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. Results: 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 Lactobacillus species. 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. Conclusion: 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.",
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