Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome
ABSTRACT The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment but also to the use of conflicting methodologies to study its microbial composition. Here, we systematically assessed best practices for the sequencing-based charact...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2020-12-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00448-20 |
| _version_ | 1819007122619236352 |
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| author | Luisa W. Hugerth Marcela Pereira Yinghua Zha Maike Seifert Vilde Kaldhusdal Fredrik Boulund Maria C. Krog Zahra Bashir Marica Hamsten Emma Fransson Henriette Svarre Nielsen Ina Schuppe-Koistinen Lars Engstrand |
| author_facet | Luisa W. Hugerth Marcela Pereira Yinghua Zha Maike Seifert Vilde Kaldhusdal Fredrik Boulund Maria C. Krog Zahra Bashir Marica Hamsten Emma Fransson Henriette Svarre Nielsen Ina Schuppe-Koistinen Lars Engstrand |
| author_sort | Luisa W. Hugerth |
| collection | DOAJ |
| description | ABSTRACT The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment but also to the use of conflicting methodologies to study its microbial composition. Here, we systematically assessed best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region performed best in silico, but limitations of current sequencing technologies meant that the V3-V4 region performed equally well. Both approaches presented very good agreement with qPCR quantification of key taxa, provided that an appropriate bioinformatic pipeline was used. Shotgun metagenomic sequencing presents an interesting alternative to 16S rRNA gene amplification and sequencing but requires deeper sequencing and more bioinformatic expertise and infrastructure. We assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and -use reference database of vaginal taxa. This curated database performed as well as the best-performing previously published strategies. Despite the many advantages of shotgun sequencing, none of the shotgun approaches assessed here agreed with the qPCR data as well as the 16S rRNA gene sequencing. IMPORTANCE The vaginal microbiome has been connected to various aspects of host health, including susceptibility to sexually transmitted infections as well as gynecological cancers and pregnancy outcomes. This has led to a thriving research environment but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions. |
| first_indexed | 2024-12-21T00:19:34Z |
| format | Article |
| id | doaj.art-615c12678b544adf99d3e8489166a079 |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-21T00:19:34Z |
| publishDate | 2020-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-615c12678b544adf99d3e8489166a0792022-12-21T19:22:07ZengAmerican Society for MicrobiologymSphere2379-50422020-12-015610.1128/mSphere.00448-20Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal MicrobiomeLuisa W. Hugerth0Marcela Pereira1Yinghua Zha2Maike Seifert3Vilde Kaldhusdal4Fredrik Boulund5Maria C. Krog6Zahra Bashir7Marica Hamsten8Emma Fransson9Henriette Svarre Nielsen10Ina Schuppe-Koistinen11Lars Engstrand12Centre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenDepartment of Medicine Solna, Division of Infectious Diseases, Karolinska University Hospital, Center for Molecular Medicine, Karolinska Institutet, Solna, SwedenCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenThe Recurrent Pregnancy Loss Unit, Capital Region of Denmark, Rigshospitalet and Hvidovre Hospital, Copenhagen, DenmarkThe Recurrent Pregnancy Loss Unit, Capital Region of Denmark, Rigshospitalet and Hvidovre Hospital, Copenhagen, DenmarkCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenThe Recurrent Pregnancy Loss Unit, Capital Region of Denmark, Rigshospitalet and Hvidovre Hospital, Copenhagen, DenmarkCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenCentre for Translational Microbiome Research, Department of Microbiology, Tumour and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Solna, SwedenABSTRACT The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment but also to the use of conflicting methodologies to study its microbial composition. Here, we systematically assessed best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region performed best in silico, but limitations of current sequencing technologies meant that the V3-V4 region performed equally well. Both approaches presented very good agreement with qPCR quantification of key taxa, provided that an appropriate bioinformatic pipeline was used. Shotgun metagenomic sequencing presents an interesting alternative to 16S rRNA gene amplification and sequencing but requires deeper sequencing and more bioinformatic expertise and infrastructure. We assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and -use reference database of vaginal taxa. This curated database performed as well as the best-performing previously published strategies. Despite the many advantages of shotgun sequencing, none of the shotgun approaches assessed here agreed with the qPCR data as well as the 16S rRNA gene sequencing. IMPORTANCE The vaginal microbiome has been connected to various aspects of host health, including susceptibility to sexually transmitted infections as well as gynecological cancers and pregnancy outcomes. This has led to a thriving research environment but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.https://journals.asm.org/doi/10.1128/mSphere.00448-2016S rRNAPCRampliconhuman microbiomemetagenomicsmolecular methods |
| spellingShingle | Luisa W. Hugerth Marcela Pereira Yinghua Zha Maike Seifert Vilde Kaldhusdal Fredrik Boulund Maria C. Krog Zahra Bashir Marica Hamsten Emma Fransson Henriette Svarre Nielsen Ina Schuppe-Koistinen Lars Engstrand Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome mSphere 16S rRNA PCR amplicon human microbiome metagenomics molecular methods |
| title | Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome |
| title_full | Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome |
| title_fullStr | Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome |
| title_full_unstemmed | Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome |
| title_short | Assessment of <italic toggle="yes">In Vitro</italic> and <italic toggle="yes">In Silico</italic> Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome |
| title_sort | assessment of italic toggle yes in vitro italic and italic toggle yes in silico italic protocols for sequence based characterization of the human vaginal microbiome |
| topic | 16S rRNA PCR amplicon human microbiome metagenomics molecular methods |
| url | https://journals.asm.org/doi/10.1128/mSphere.00448-20 |
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