The profiling of microbiota in vaginal swab samples using 16S rRNA gene sequencing and IS-pro analysis

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Abstract

Background

16S rRNA gene sequencing is currently the most common way of determining the composition of microbiota. This technique has enabled many new discoveries to be made regarding the relevance of microbiota to the health and disease of the host. However, compared to other diagnostic techniques, 16S rRNA gene sequencing is fairly costly and labor intensive, leaving room for other techniques to improve on these aspects.

Results

The current study aimed to compare the output of 16S rRNA gene sequencing to the output of the quick IS-pro analysis, using vaginal swab samples from 297 women of reproductive age. 16S rRNA gene sequencing and IS-pro analyses yielded very similar vaginal microbiome profiles, with a median Pearson’s R ² of 0.97, indicating a high level of similarity between both techniques.

Conclusions

We conclude that the results of 16S rRNA gene sequencing and IS-pro are highly comparable and that both can be used to accurately determine the vaginal microbiota composition, with the IS-pro analysis having the benefit of rapidity.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-021-02149-7.

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Most cited references 16

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Search and clustering orders of magnitude faster than BLAST.

Robert Edgar (2010)

Biological sequence data is accumulating rapidly, motivating the development of improved high-throughput methods for sequence classification. UBLAST and USEARCH are new algorithms enabling sensitive local and global search of large sequence databases at exceptionally high speeds. They are often orders of magnitude faster than BLAST in practical applications, though sensitivity to distant protein relationships is lower. UCLUST is a new clustering method that exploits USEARCH to assign sequences to clusters. UCLUST offers several advantages over the widely used program CD-HIT, including higher speed, lower memory use, improved sensitivity, clustering at lower identities and classification of much larger datasets. Binaries are available at no charge for non-commercial use at http://www.drive5.com/usearch.

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Vaginal microbiome of reproductive-age women.

Jacques Ravel, Pawel Gajer, Zaid Abdo … (2011)

The means by which vaginal microbiomes help prevent urogenital diseases in women and maintain health are poorly understood. To gain insight into this, the vaginal bacterial communities of 396 asymptomatic North American women who represented four ethnic groups (white, black, Hispanic, and Asian) were sampled and the species composition characterized by pyrosequencing of barcoded 16S rRNA genes. The communities clustered into five groups: four were dominated by Lactobacillus iners, L. crispatus, L. gasseri, or L. jensenii, whereas the fifth had lower proportions of lactic acid bacteria and higher proportions of strictly anaerobic organisms, indicating that a potential key ecological function, the production of lactic acid, seems to be conserved in all communities. The proportions of each community group varied among the four ethnic groups, and these differences were statistically significant [χ(2)(10) = 36.8, P < 0.0001]. Moreover, the vaginal pH of women in different ethnic groups also differed and was higher in Hispanic (pH 5.0 ± 0.59) and black (pH 4.7 ± 1.04) women as compared with Asian (pH 4.4 ± 0.59) and white (pH 4.2 ± 0.3) women. Phylotypes with correlated relative abundances were found in all communities, and these patterns were associated with either high or low Nugent scores, which are used as a factor for the diagnosis of bacterial vaginosis. The inherent differences within and between women in different ethnic groups strongly argues for a more refined definition of the kinds of bacterial communities normally found in healthy women and the need to appreciate differences between individuals so they can be taken into account in risk assessment and disease diagnosis.

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An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform

Douglas Fadrosh, Bing Ma, Pawel Gajer … (2014)

Background To take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality. Results Here we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp “heterogeneity spacer” to the index sequence that allows an equal proportion of samples to be sequenced out of phase. Conclusions Our approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.

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Author and article information

Contributors

R. Koedooder: r.koedooder@erasmusmc.nl

Journal

Journal ID (nlm-ta): BMC Microbiol

Journal ID (iso-abbrev): BMC Microbiol

Title: BMC Microbiology

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2180

Publication date (Electronic): 31 March 2021

Publication date PMC-release: 31 March 2021

Publication date Collection: 2021

Volume: 21

Electronic Location Identifier: 100

Affiliations

[1 ]Laboratory of Immunogenetics, Department of Medical Microbiology and Infection Control, Amsterdam UMC, location VUmc, Amsterdam, the Netherlands

[2 ]Tubascan, Spin-off at the Department of Medical Microbiology and Infection Control, Amsterdam UMC, location VUmc, Amsterdam, the Netherlands

[3 ]GRID grid.5645.2, ISNI 000000040459992X, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, , Erasmus University Medical Center, ; Wytemaweg 12, Rotterdam, 3015 CN The Netherlands

[4 ]InBiome B.V, Amsterdam, The Netherlands

[5 ]GRID grid.5645.2, ISNI 000000040459992X, Division Obstetrics, Department of Obstetrics and Gynecology, , Erasmus University Medical Centre, ; Rotterdam, The Netherlands

[6 ]GRID grid.412966.e, ISNI 0000 0004 0480 1382, Department of Medical Microbiology, , Maastricht University Medical Center, ; Maastricht, the Netherlands

[7 ]Department of Medical Microbiology and Infection Control, Amsterdam UMC, location VUmc, Amsterdam, the Netherlands

[8 ]ARTPred B.V, Seringenstraat 15, ‘s Hertogenbosch, 5213 GS The Netherlands

[9 ]GRID grid.5012.6, ISNI 0000 0001 0481 6099, Institute of Public Health Genomics, Department of Genetics and Cell Biology, Research Institute GROW, Faculty of Health, Medicine & Life Sciences, , University of Maastricht, ; Maastricht, The Netherlands

Article

Publisher ID: 2149

DOI: 10.1186/s12866-021-02149-7

PMC ID: 8015044

PubMed ID: 33789573

SO-VID: afa248f6-7396-4839-90f7-a741b3df7d77

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 30 September 2020

Date accepted : 29 January 2021

Custom metadata

ScienceOpen disciplines: Microbiology & Virology

Keywords: vaginal microbiota,is-pro,16s rrna gene sequencing

Data availability:

ScienceOpen disciplines: Microbiology & Virology

Keywords: vaginal microbiota, is-pro, 16s rrna gene sequencing

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The profiling of microbiota in vaginal swab samples using 16S rRNA gene sequencing and IS-pro analysis

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Abstract

Background

Results

Conclusions

Supplementary Information

Related collections

Tick microbiome

Most cited references 16

Search and clustering orders of magnitude faster than BLAST.

Vaginal microbiome of reproductive-age women.

An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform

Author and article information

Contributors

Journal

Affiliations

Article

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Cited by 5

Most referenced authors 387