Open AccessComparative analysis of DNA extraction and PCR product purification methods for cervicovaginal microbiome analysis using cpn60 microbial profiling
Author(s) -
Elinor Shvartsman,
Meika Richmond,
John J. Schellenberg,
Alana Lamont,
Cátia T. Perciani,
Justen Russell,
Vanessa Poliquin,
Adam Burgener,
Walter Jaoko,
Paul Sandstrom,
Kelly S. MacDonald
Publication year - 2022
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0262355
Subject(s) - dna extraction , biology , amplicon , polymerase chain reaction , lysis , microbiome , dna , chromatography , microbiology and biotechnology , chemistry , genetics , gene
Background The microbiota of the lower female genital tract plays an important role in women’s health. Microbial profiling using the chaperonin 60 ( cpn 60) universal target (UT) improves resolution of vaginal species associated with negative health outcomes compared to the more commonly used 16S ribosomal DNA target. However, the choice of DNA extraction and PCR product purification methods may bias sequencing-based microbial studies and should be optimized for the sample type and molecular target used. In this study, we compared two commercial DNA extraction kits and two commercial PCR product purification kits for the microbial profiling of cervicovaginal samples using the cpn 60 UT. Methods DNA from cervicovaginal secretions and vaginal lavage samples as well as mock community standards were extracted using either the specialized QIAamp DNA Microbiome Kit, or the standard DNeasy Blood & Tissue kit with enzymatic pre-treatment for enhanced lysis of gram-positive bacteria. Extracts were PCR amplified using well-established cpn 60 primer sets and conditions. Products were then purified using a column-based method (QIAquick PCR Purification Kit) or a gel-based PCR clean-up method using the QIAEX II Gel Extraction Kit. Purified amplicons were sequenced with the MiSeq platform using standard procedures. The overall quality of each method was evaluated by measuring DNA yield, alpha diversity, and microbial composition. Results DNA extracted from cervicovaginal samples using the DNeasy Blood and Tissue kit, pre-treated with lysozyme and mutanolysin, resulted in increased DNA yield, bacterial diversity, and species representation compared to the QIAamp DNA Microbiome kit. The column-based PCR product purification approach also resulted in greater average DNA yield and wider species representation compared to a gel-based clean-up method. In conclusion, this study presents a fast, effective sample preparation method for high resolution cpn 60 based microbial profiling of cervicovaginal samples.