Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota

Gill, A. Christina; Wijgert, Janneke van de; Blow, Frances; Darby, Alistair C.

doi:10.1371/journal.pone.0163148

Cited by 73 publications

(66 citation statements)

References 64 publications

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“…Hence, biological variability of the vaginal microbiota exceeds the extent of technical variation rising from the sampling collection technique (this study) and DNA extraction method [21]. Therefore past and future vaginal microbiota studies employing different sampling strategies should be comparable in the absence of other technical confounders, such as sample storage, sequencing strategy or bioinformatic parameters.…”

Section: Discussionmentioning

confidence: 99%

Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

et al. 2017

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BackgroundMolecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species.ResultsThe total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001).ConclusionsThe results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal microbiota studies employing different sampling strategies should be comparable in the absence of other technical confounders. The Evalyn® self-sampling device performed equally well compared to samples taken by a clinician, and hence offers a good-quality microbiota sample without the need for a gynecological examination. The amount of collected sample as well as the DNA and protein yield varied across the sampling techniques, which may have practical implications for study design.

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Section: Discussionmentioning

confidence: 99%

Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

et al. 2017

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“…It is currently not known how rectovaginal and vaginal selective cultures, PCR, and sequencing results relate to one another, but it is possible that pathobionts are not only under-detected in sequencing studies due to the bioinformatics used, but also due to DNA extraction, amplification, and other biases. For example, the detection rate in the HARP study was especially low, which may have been due to the fact that we did not use bead-beating during DNA extraction in that study (Gill et al, 2016). Third, some of the standard diagnostic tests that we used are known to have lower sensitivity than NAAT-based tests (e.g., culture for vulvovaginal candidiasis and T. vaginalis), and not all women in all three studies were screened for all STI pathogens.…”

Section: Pathobionts In the Vaginal Microbiotamentioning

confidence: 92%

Pathobionts in the Vaginal Microbiota: Individual Participant Data Meta-Analysis of Three Sequencing Studies

Wijgert

Verwijs

Gill

et al. 2020

Front. Cell. Infect. Microbiol.

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Having pathobionts was positively associated with young age, non-Dutch origin, hormonal contraceptive use, smoking, antibiotic use in the 14 days prior to sampling, van de Wijgert et al. Pathobionts in the Vaginal MicrobiotaHIV status, and the presence of sexually transmitted pathogens, in at least one but not all studies; inconsistently associated with sexual risk-taking and unusual vaginal discharge reporting; and not associated with vaginal yeasts detection by microscopy. We recommend that future VMB studies quantify common vaginal pathobiont genera.

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“…Sample collection, DNA extraction, and PCR primers impact sensitivity to different taxa (17,18), sometimes so strongly that particular taxa can go undetected (e.g., Gardnerella; ref. 19).…”

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Replication and refinement of a vaginal microbial signature of preterm birth in two racially distinct cohorts of US women

Callahan

DiGiulio

Goltsman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

327

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Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. Previous studies have suggested that the maternal vaginal microbiota contributes to the pathophysiology of PTB, but conflicting results in recent years have raised doubts. We conducted a study of PTB compared with term birth in two cohorts of pregnant women: one predominantly Caucasian (n = 39) at low risk for PTB, the second predominantly African American and at high-risk (n = 96). We profiled the taxonomic composition of 2,179 vaginal swabs collected prospectively and weekly during gestation using 16S rRNA gene sequencing. Previously proposed associations between PTB and lower Lactobacillus and higher Gardnerella abundances replicated in the low-risk cohort, but not in the high-risk cohort. High-resolution bioinformatics enabled taxonomic assignment to the species and subspecies levels, revealing that Lactobacillus crispatus was associated with low risk of PTB in both cohorts, while Lactobacillus iners was not, and that a subspecies clade of Gardnerella vaginalis explained the genus association with PTB. Patterns of cooccurrence between L. crispatus and Gardnerella were highly exclusive, while Gardnerella and L. iners often coexisted at high frequencies. We argue that the vaginal microbiota is better represented by the quantitative frequencies of these key taxa than by classifying communities into five community state types. Our findings extend and corroborate the association between the vaginal microbiota and PTB, demonstrate the benefits of high-resolution statistical bioinformatics in clinical microbiome studies, and suggest that previous conflicting results may reflect the different risk profile of women of black race.pregnancy | prematurity | vaginal microbiota | Lactobacillus | Gardnerella P reterm birth (PTB; delivery at <37 gestational wk) affects ≈12% of US births and is the leading cause of neonatal death and morbidity worldwide. Multiple lines of evidence support a role for the indigenous microbial communities of the mother (the maternal microbiota) in the pathophysiology of PTB. Microbial invasion of the amniotic cavity is one of the most frequent causes of spontaneous PTB (1), and the most common invading taxa are consistent with maternal origin (2-4). Bacterial vaginosis (BV), a condition involving an altered vaginal microbiota, has been consistently identified as a risk factor for PTB (5, 6). Multiple studies have also found chronic periodontitis, another condition associated with an altered microbiota, to be a risk factor for PTB (7,8).High-throughput sequencing methods have facilitated new lines of investigation into the microbial etiology of PTB (9, 10). Amplification and high-throughput sequencing of the 16S rRNA gene (metabarcoding) simultaneously measures the presence and relative abundance of thousands of bacterial taxa (composition), and resolves differences to the level of genus and sometimes species or subspecies. To date, metabarcoding studies of the relationship between the vaginal microbiota and PTB have y...

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Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota

Cited by 73 publications

References 64 publications

Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

Pathobionts in the Vaginal Microbiota: Individual Participant Data Meta-Analysis of Three Sequencing Studies

Replication and refinement of a vaginal microbial signature of preterm birth in two racially distinct cohorts of US women

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