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

Takeo Shibata,William W Greenfield,Sarah M Owens,Hannah N Coleman,Michael S Robeson,Mayumi Nakagawa,Toshiyuki Sasagawa,Brenda A Wilson

doi:10.1371/journal.pone.0237556

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.

Highlights

High-throughput sequencing (HTS) technology of 16S rRNA gene amplicon sequences has made it possible to better understand the relationships between cervicovaginal microbiota and human papillomavirus (HPV) infection [1,2,3,4,5] and HPV-related diseases [6,7,8,9,10]
Observed differences in microbial composition were due to the significant influence of the individual patient and not the extraction protocol
We have shown that the ability to characterize cervical microbiota from liquid-based cytology (LBC) specimens is possible, we were limited in our ability to directly assess if the observed microbial community composition would reflect that of a fresh sample

Summary

Introduction

High-throughput sequencing (HTS) technology of 16S rRNA gene amplicon sequences has made it possible to better understand the relationships between cervicovaginal microbiota and human papillomavirus (HPV) infection [1,2,3,4,5] and HPV-related diseases [6,7,8,9,10]. Cervicovaginal microbiota are considered to be an important factor affecting the progress of cervical.

Methods

Results

Discussion

Conclusion