RT Journal Article
SR Electronic
T1 Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.01.27.921619
DO 10.1101/2020.01.27.921619
A1 Takeo Shibata
A1 Mayumi Nakagawa
A1 Hannah N. Coleman
A1 Sarah M. Owens
A1 William W. Greenfield
A1 Toshiyuki Sasagawa
A1 Michael S. Robeson II
YR 2021
UL http://biorxiv.org/content/early/2021/07/19/2020.01.27.921619.abstract
AB 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 StatementM.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.