<p class="first" id="P3">Metagenomic sequencing of bacterial samples has become the
gold standard for profiling
microbial populations, but 16S rRNA profiling remains widely used due to advantages
in sample throughput, cost, and sensitivity even though the approach is hampered by
primer bias and lack of specificity. We hypothesized that a hybrid approach, that
combined targeted PCR amplification with high-throughput sequencing of multiple regions
of the genome, would capture many of the advantages of both approaches. We developed
a method that identifies and quantifies members of bacterial communities through simultaneous
analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines
high-throughput microfluidics for PCR amplification, short read DNA sequencing, and
a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON)
for optimizing taxonomic assignment. MVRSION performance was compared to single variable
region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains
using existing software (QIIME), and the results of community profiling by shotgun
sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized
with a defined, phylogenetically diverse consortium of human gut bacterial strains.
Positive predictive values for MVSION ranged from 65%−91% versus 44%−61% for single
region QIIME analyses (p<0.01, p<0.001), while the abundance estimate r
<sup>2</sup> for MVRSION compared to COPRO-Seq was 0.77 vs. 0.46 and 0.45 for V3-QIIME
and V4-QIIME,
respectively. MVRSION represents a generally applicable tool for taxonomic classification
that is superior to singleregion 16S rRNA methods, resource efficient, highly scalable
for assessing the microbial composition of up to thousands of samples concurrently,
with multiple applications ranging from whole community profiling to targeted tracking
of organisms of interest in diverse habitats as a function of specified variables/perturbations.
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