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Comparison of Fecal Collection Methods on Variation in Gut Metagenomics and Untargeted Metabolomics.

Author(s): Guan H, Pu Y, Liu C, Lou T, Tan S, Kong M, Sun Z, Mei Z, Qi Q, Quan Z, Zhao G, Zheng Y

Publication: mSphere, 2021, Vol. 6, Page e0063621

PubMed ID: 34523982 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the metagenome analyzed) of fecal specimens from healthy volunteers that were stored in 95% ethanol, RNAlater, OMNIgene GUT, FOBT cards, or Microlution for 0 or 4 days with matched specimens stored frozen at -80°C using whole genome sequencing (WGS). Fecal specimens were self-collected from eight healthy volunteers (six male and two female) and manually mixed with a spatula. Each specimen was divided into eleven aliquots. One aliquot was placed in a feces tube (no preservative) and frozen immediately at -80°C (control) and two aliquots each were placed into tubes containing 500 µL 95% ethanol, tubes containing 500 µL RNAlater and 200 mg zirconia/silica beads, Microlution tubes, or OMNIgene GUT tubes and immediately mixed by shaking. The remaining two aliquots were smeared onto fecal occult blood test [FOBT] cards. One specimen for each preservative was frozen within 30 min of collection and stored at -80°C while the second was stored for 4 days at room temperature before freezing. Specimens were thawed on ice, after which a portion of each specimen was placed in an Eppendorf tube and DNA was extracted using the TIANamp stool DNA Kit. DNA was quantified using the Equalbit -stranded DNA (dsDNA) High-Sensitivity (HS) Assay kit. Sequencing libraries were constructed using the Tn5 DNA Library Prep Kit and pair-end sequenced on a Novaseq6000 instrument.  After removal of reads mapping to the human genome, the taxonomic profiles were evaluated using MetaPhlan3. 

   Summary of Findings:

   Principal-coordinate analysis (PCoA) based on Bray-Curtis distances of the microbiome showed clustering based on patient source, but no clear clustering based on preservative or storage duration was observed. Most of the variability in the microbiome was attributed to interindividual variability (77%), with a much lower percentage of the variability explained by preservative (3.6%) or storage duration (2.6%). The stability of the top four most abundant phyla and microbial diversity at the gene and species level were dependent on the preservative used, with the highest intraclass correlation coefficients (ICC) between the two timepoints (day 0 and 4) generally observed in specimens preserved using OMNIgene GUT (≥0.75, except for actinobacteria) and the lowest in specimens preserved using 95% ethanol. The ICC for the two α-diversity metrics (Shannon and Simpson) and richness at the species and gene level were affected by preservative type (ICC 0.20-0.75 for 95% ethanol, 0.20-0.80 for RNAlater, ≥0.40 for all other preservatives), but the β-diversity measures (Bray-Curtis distance and Jaccard distance) at the species and gene level showed high stability (ICC ≥0.75) for all preservatives between the timepoints. The stability at the pathway level between timepoints was high (ICCs ≥0.74) for all diversity metrics for specimens preserved in OMNIgene GUT, on FOBT cards, or in RNAlater.  Further analysis showed the relative abundance of top four most abundant phyla were stable between timepoints in specimens stored in Microlution, OMNIgene GUT, or on FOBT cards, but not in 95% ethanol or RNAlater. The 20 most abundant pathways were generally less affected by 4 days of storage when specimens were preserved with OMNIgene GUT, FOBT cards, and RNAlater than Microlution or 95% ethanol. Importability, levels of four genes associated with antibiotic resistance were affected by specimen storage for 4 days in 95% ethanol. When specimens stored for 4 days with preservative were compared to the control (immediately frozen without preservative), the ICC showed high reliability (ICC ≥0.75) in all metrics at the species level (α and β-diversity, richness) for specimens stored in OMNIgene GUT and on FOBT cards. Interestingly, at the gene level, the ICC for both α -diversity indices between specimens stored for 4 days with preservative and control specimens was low (most <0.4) for all preservatives. Nevertheless, specimens stored for 4 days with OMNIgene GUT, FOBT cards, or Microlution had similar relative abundances of the top four most abundant phyla, and microbial diversity indices at the species, gene, and pathway levels to those in control specimens. Among specimens stored for 4 days in preservatives, pathway abundance was most similar to control specimens when specimens were preserved with FOBT cards followed by OMNIgene GUT, RNAlater, and Microlution. A total of 14 pathways were found at lower levels than in control specimens after specimens were stored for 4 days in 95% ethanol.  The 20 most abundant genes associated with antibiotic resistance were least affected by storage when specimens were preserved with Microlution and most affected by storage when storage was in 95% ethanol.  Among specimens stored for 4 days, the overlap in detection of the 21 species involved in short chain fatty acid (SCFA) production and the 9 genera involved in secondary bile acid (SBA) metabolism in the control specimen was highest for FOBT cards (16 species and 6 genera), followed by OMNIgene GUT (16 species and 4 genera), Microlution (14 species and 4 genera), RNAlater (15 species and 3 genera), and 95% ethanol (8 species and 2 genera). The authors conclude that OMNIgene GUT, FOBT cards, RNAlater, and Microlution, but not 95% ethanol, are suitable for fecal metagenomic studies. 

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen
   • Ethanol
   • RNAlater
   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 days 4 days
   Biospecimen Preservation	Type of fixation/preservation	FOBT Card Ethanol Frozen RNAlater OMNIgene GUT Microlution

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2. Study Purpose

   This study compared the metabolome of fecal specimens that were stored frozen at -80°C with matched specimens that were stored in 95% ethanol, RNAlater, OMNIgene GUT, FOBT cards, or Microlution for 0 or 4 days using untargeted ultra high-performance liquid chromatography (UHPLC). Fecal specimens were self-collected from eight healthy volunteers (six male and two female) and manually mixed with a spatula and aliquoted. One aliquot was placed in a feces tube (no preservative) and frozen immediately at -80°C (control) and two aliquots each were placed into tubes containing 500 µL 95% ethanol, tubes containing 500 µL RNAlater and 200 mg beads, Microlution tubes, or OMNIgene GUT tubes and immediately mixed by shaking. One specimen for each preservative was frozen within 30 min of collection and stored at -80°C while the second was stored for 4 days at room temperature before freezing. Specimens were thawed on ice, freeze-dried, and resuspended in methanol. The metabolome was analyzed by ultra high-performance liquid chromatography (UHPLC). The XCMS package in R was used to identify peaks and correct retention time, after which features were matched using the HMDB database. 

   Summary of Findings:

   Principal-component analysis (PCA) based on the metabolome showed clustering based on patient source but no clear clustering based on preservative or storage duration. Most of the variability in the metabolome was attributed to interindividual variability (37%), with a much lower percentage of the variability explained by preservative (4.9%) and storage duration (0.8%). Depending on the preservation method, 1929-1992 metabolites were identified, with 99.5%-99.8% of the metabolites detected in the control specimen detected in specimens preserved with each preservative. The highest overlap in metabolite detection with the control was observed for specimens preserved with 95% ethanol (overall 99.8%; 99.4%, 99.0%, and 95.8% for metabolites detected in ≥50%, ≥80%, and 100% of specimens, respectively), followed by Microlution (overall 99.6%; 97.6%, 97.0%, and 90.4% for metabolites detected in ≥50%, ≥80%, and 100% of specimens, respectively), with lower overlaps detected for RNAlater (overall 99.5%; 96.3%, 92.6%, and 68.1% for metabolites detected in ≥50%, ≥80%, and 100% of specimens, respectively) and OMNIgene GUT (overall 96.7%; 90.6%, 87.0%, and 78.9% for metabolites detected in ≥50%, ≥80%, and 100% of specimens, respectively). The highest ICC with control specimens for amino acids, lipids, nucleotides, and peptides was observed in specimens preserved with 95% ethanol, followed by Microlution. The highest ICC with control specimens for carbohydrates, cofactors and vitamins, and xenobiotics was observed when fecal specimens were preserved with Microlution, followed by 95% ethanol. The ICC in levels of metabolites detected in ≥80% of specimens were highest between the two time-points and between the control and specimens preserved in 95% ethanol at either day 0 or day 4 of storage. Each of the 5 short chain fatty acids (SCFAs) and 8 secondary bile acids (SBAs) that were detected in the control specimen were found in specimens preserved in 95% ethanol. The authors conclude that 95% ethanol is the best preservative for metabolomic studies. 

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Ethanol
   • RNAlater
   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Small molecule	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 days 4 days
   Biospecimen Preservation	Type of fixation/preservation	Ethanol Frozen FineFIX OMNIgene GUT RNAlater Microlution

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