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Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota

View ORCID ProfileTakeo Shibata, View ORCID ProfileMayumi Nakagawa, Hannah N. Coleman, View ORCID ProfileSarah M. Owens, William W. Greenfield, View ORCID ProfileToshiyuki Sasagawa, View ORCID ProfileMichael S. Robeson II
doi: https://doi.org/10.1101/2020.01.27.921619
Takeo Shibata
1Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
2Department of Obstetrics and Gynecology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
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Mayumi Nakagawa
1Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
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Hannah N. Coleman
1Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
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Sarah M. Owens
3Biosciences Division, Argonne National Laboratory, Lemont, IL, USA
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William W. Greenfield
4Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
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Toshiyuki Sasagawa
2Department of Obstetrics and Gynecology, Kanazawa Medical University, Uchinada, Ishikawa, Japan
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Michael S. Robeson II
5Department of Biomedical Informatics, University of Arkansas for Medical Sciences, Little Rock, AR, USA
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  • ORCID record for Michael S. Robeson II
  • For correspondence: MRobeson@uams.edu
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Abstract

Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 ± 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.

Competing Interest Statement

M.N. is one of the inventors named in the patents and patent applications for the HPV therapeutic vaccine PepCan. The remaining authors declare no conflicts of interest.

Footnotes

  • We've received additional helpful comments to increase the clarity of our work. In particular we've added post-hoc testing to some of our analyses, and associated figures. We've further emphasized the limitations of our work, and focused on the comparative aspect, i.e. the accessibility of microbial DNA between the extraction kits. This resulted in the addition and modification of text and figures. The overall content and perspectives of the manuscript remains unchanged. Some of the alterations and formatting had to do with meeting other journal requirements.

  • https://www.ncbi.nlm.nih.gov/bioproject/PRJNA598197

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 19, 2021.
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Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota
Takeo Shibata, Mayumi Nakagawa, Hannah N. Coleman, Sarah M. Owens, William W. Greenfield, Toshiyuki Sasagawa, Michael S. Robeson II
bioRxiv 2020.01.27.921619; doi: https://doi.org/10.1101/2020.01.27.921619
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Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota
Takeo Shibata, Mayumi Nakagawa, Hannah N. Coleman, Sarah M. Owens, William W. Greenfield, Toshiyuki Sasagawa, Michael S. Robeson II
bioRxiv 2020.01.27.921619; doi: https://doi.org/10.1101/2020.01.27.921619

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