Methodologic differences and sources of bias
There are known sources of bias in metagenomic studies associated with
analysis methods.2,12,13 Brooks and colleagues
determined that microbiome community composition can be biased at
various steps including DNA extraction, PCR amplification, sequencing,
and taxonomic classification.12 The researchers
reported that more than 85% of the microbiome community results were
biased by small variations in methods (less than 5%). Specifically, it
has been determined that variations in DNA extraction and amplification
can result in decreased representation of Streptococcus species.
If women with GBS colonization only harbor small amounts of the
bacteria, it is possible that bias introduced by these steps could have
contributed to the limited representation of GBS in vaginal microbiome
data.
Such underrepresentation of known pathogens can significantly limit the
clinical scope of vaginal microbiome studies. For example, many studies
only reported results related to community state types or analyzed the
most abundant species. Consequently, if thresholds for inclusion are
based on sample abundance (e.g., 5% of sample or 5,000 reads), GBS data
may have been excluded if it did not meet the threshold.
Furthermore, selection of 16S regions for amplification and associated
primers can influence genus and species level
resolution.14 For example, it is generally more
difficult to distinguish Lactobacillus species within the V4
region, but may be easier to do so in V1-V3.15Conversely, G. vaginalis is more difficult to detect and
differentiate within the V1-V2 region because its sequence is highly
variable prior to the V1 region, making primer selection more
complex.14 These biases can be partially mitigated by
recognizing the characteristics of 16S rRNA sequencing and thoughtfully
selecting primers. Unfortunately, studies that evaluate the influence of
selected 16S regions on GBS representation have not been published yet.
From our findings, the 13 studies that detected GBS used 16S regions
between V1-V4 or V6. However, the 32 studies that did not report GBS
also used the same regions. Thus, assessment of bias by 16S region is
important when designing and evaluating future vaginal microbiome
studies.
Given limitations in exploring the true proportions within the microbial
community, studies have increasingly employed whole genome sequencing
(WGS), which allows for higher resolution up to the strain level. In our
review, two studies employed WGS but did not report
GBS.16,17 Both papers initially selected some of the
most frequent species-level taxa, which are frequentlyLactobacillus and Gardnerella species, to continue further
analysis. This approach may have constrained the strength of WGS,
causing researchers to miss the rarer, yet clinically important genus,
species, and strains.