Effects of Variation in Urine Sample Storage Conditions on 16S Urogenital Microbiome Analyses.
Author(s): Kumar T, Bryant M, Cantrell K, Song SJ, McDonald D, Tubb HM, Farmer S, Lukacz ES, Brubaker L, Knight R
Publication: mSystems, 2022, Vol. , Page e0102922
PubMed ID: 36475896 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare the alpha and beta diversity of unpreserved frozen urine, and urine preserved with GeneLock that was stored at various temperature with freeze-thaw or heat cycling.
Conclusion of Paper
Both beta diversity and alpha diversity were primarily affected by patient source, although a small but statistically significant effect of storage temperature on alpha-diversity was identified. Among specimens frozen at -20°C, a small difference was found between specimens preserved with GeneLock and those that were not. The authors conclude that bias introduced in the microbiome by the use of Genelock as a urine preservative and by extreme storage temperatures is small by comparison to inter-individual variation.
Studies
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Study Purpose
The purpose of this study was to compare the alpha and beta diversity of unpreserved frozen urine, and urine preserved with GeneLock that was stored at various temperature with freeze-thaw or heat cycling. Urine was collected from ten healthy volunteers and immediately aliquoted into tubes with and without Genelock preservative (one to ten). Aliquots with and without Genelock were stored at -20°C for 48 h, additional aliquots with Genelock were stored at room temperature for 48 h, at 4°C with and without 2 warming cycles to 40°C, and at -20°C with two thaw cycles at room temperature. Urine was then plated, and RNA was extracted using Earth Microbiome Project protocols and then sequenced on an Illumina MiSeq.
Summary of Findings:
Weighted and unweighted UniFrac, permutational multivariate analysis of variance (PERMANOVA) revealed that beta diversity was significantly affected by patient source (PERMANOVA, P = 0.001, both), but not preservative method (PERMANOVA, P = 0.43 and P = 0.96, respectively) or storage temperature (PERMANOVA, unweighted P = 0.25 and P = 0.76, respectively). The clustering of the weighted UniFrac was much tighter than the unweighted UniFrac, which the authors state indicates that clustering was driven by similarities rather than rare taxa. The mean difference in alpha diversity was close to zero between unpreserved urine and urine preserved with GeneLock that was stored at the various temperatures specified above with and without freeze-thaw cycling, indicating that alpha diversity is primarily based on patient source rather than storage temperature. While inter-individual variation accounted for the majority of variation (Jaccard [R2 = 0.81], unweighted UniFrac [R2 = 0.76], weighted UniFrac [R2 = 0.72], and BrayCurtis [R2 = 0.92]), a small but statistically significant effect of storage temperature was also identified (Jaccard [R2 = 0.017], unweighted UniFrac [R2 = 0.012], weighted UniFrac [R2 = 0.037], and Bray-Curtis [R2 = 0.05]). In specimens frozen at -20°C, a small difference was found between specimens preserved with GeneLock and those that were not. The authors conclude that bias introduced in the microbiome by the use of Genelock as a urine preservative and by extreme storage temperatures is small by comparison to inter-individual variation.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Short-term storage solution None
GeneLock
Storage Storage temperature -20°C
4°C
Room temperature
40°C
Storage Freeze/thaw cycling 0 cycles
2 cycles