Fig 1.
Schematic representation of the DNA extraction and PCR product purification methods compared in our study.
UT, universal target.
Table 1.
cpn60 PCR primers used in this investigation.
Fig 2.
Comparison of total double stranded DNA (dsDNA) yield per sample following DNA extractions as measured using the Qubit 2.0 fluorometer.
(A) Overall DNA yield per sample and replicates, (B) Paired DNA yield comparisons per extraction method used. DM, QIAamp DNA Microbiome kit, BT, modified DNeasy Blood and Tissue kit. ****P < 0.0001.
Fig 3.
Comparison of total double stranded DNA (dsDNA) yield per sample following PCR product purifications as measured using the Qubit 2.0 fluorometer using either gel-based clean-up (QIAEX II) or spin columns (QIAquick).
(A) Overall DNA yield per sample, (B) Paired comparison per purification method used. DM, QIAamp DNA Microbiome kit, BT, modified DNeasy Blood and Tissue kit protocol. NS, non-significant.
Fig 4.
Stacked bar plot comparing output of mock community standards between the different DNA extraction and PCR product purification methods.
(A) Manufacturer-extracted DNA from mock community standards, (B) Mock community standards extracted using the DM/BT kits. DM, QIAamp DNA Microbiome kit, BT, modified DNeasy Blood and Tissue kit protocol. * indicates sample had low sequencing depth (less than 20 total microbial reads). Column: refers to spin-column purification approach (QIAquick PCR purification kit). Gel: refers to gel-based PCR purification (QIAEX II kit). Aliquot numbers refer to specific extraction or PCR purification method replicates, and the follow up letters indicate a sequencing duplicate of that specific replicate.
Fig 5.
Paired comparisons of sequencing read output and microbial diversity of cervicovaginal secretion and lavage samples processed using two different DNA extraction kits.
(A) Microbial read output comparison between DNA extraction methods, (B) Observed diversity representing total different microbial species compared between the two extraction methods, (C) Shannon diversity index compared between the two extraction methods, (D) Paired host (non-microbial eukaryotes) coverage comparison between DNA extraction methods. Note that the measures had been averaged per MiSeq run duplicates. Error bars represent the standard error of the mean, DM, QIAamp DNA Microbiome kit, BT, modified DNeasy Blood and Tissue kit protocol. *P<0.05, **P < 0.01, NS, non-significant.
Fig 6.
Stacked bar plots representing the relative abundance of bacterial species and the corresponding Shannon diversity for vaginal samples processed using different DNA extraction and PCR product purification methods.
(A) Microbial profiles from VZV022V02 cervicovaginal pellet sample, (B) Microbial profiles from VZV022V03 cervicovaginal pellet sample, (C) Microbial profiles from the VM cervicovaginal lavage sample, DM, QIAamp DNA Microbiome kit, BT, modified DNeasy blood and tissue kit protocol. Only the 15 most prevalent species based on average relative abundance across samples are individually shown, with the remaining binned into the “other species” category.