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Updated: Jun 8, 2026
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Impact of sample processing method and volume on 16 S rRNA profiling of the urobiome
Sophie C Ramirez1, Zachary J Lewis2, Vanessa L Hale3
1University of Minnesota, College of Veterinary Medicine, Veterinary Medical Center, 1352 Boyd Avenue, Saint Paul, MN, 55108, USA.
Background:
The urinary microbiome (urobiome) plays important roles in both human and animal urogenital tract health. Characterization of these microbial communities presents several technical challenges, largely due to the low microbial biomass of urine. Whereas other low biomass liquid systems, such as aquatic samples, frequently employ small-pore vacuum filtration for microbial DNA concentration, urobiome studies have traditionally relied on centrifugation and pelleting of smaller volumes. Therefore, this study compared the effects of processing method (vacuum filtration versus pelleting) and sample volume on bacterial DNA yield, contaminant burden, and microbial diversity in canine urine.
Results:
A total of 50 urine aliquots were obtained across samples. Urine from 15 healthy dogs was pooled into five unique batches and divided into duplicate aliquots at volumes of 1, 3, 10, 30, and 50 mL. One aliquot was pelleted and one filtered (0.2 μm pore filter) prior to DNA extraction of the pellet or filter, respectively, and 16 S rRNA gene (V4) sequencing. Three aliquots ≥ 30 mL could not be filtered due to clogging. Sequence depth, DNA recovery, contaminant abundance, and microbial diversity were similar across urine volumes. Filtered samples contained a higher proportion of reads classified as contaminants (𝑃 = 0.002). Although beta diversity differed between methods (Bray-Curtis PERMANOVA, P = 0.007), the effect size was small (R2 = 0.03) relative to the influence of urine batch (𝑃 = 0.001, R2 = 0.65).
Conclusions:
These findings indicate that interindividual variation predominates over methodological effects. Higher urine volumes (≥ 30 mL) were associated with technical challenges in filtered samples, whereas moderate urine volumes (1-10 mL) appear sufficient for urobiome characterization. Similar microbial recovery, increased contaminant signal, and occasional clogging with filtration suggests that pelleting remains an appropriate approach for urobiome characterization.
