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Human Stool Preservation Impacts Taxonomic Profiles in 16S Metagenomics Studies.

Author(s): Plauzolles A, Toumi E, Bonnet M, Pénaranda G, Bidaut G, Chiche L, Allardet-Servent J, Retornaz F, Goutorbe B, Halfon P

Publication: Front Cell Infect Microbiol, 2022, Vol. 12, Page 722886

PubMed ID: 35211421 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared microbial DNA yields and profiles in matched aliquots of fecal specimens that were immediately frozen or stored for 14 days at fluctuating temperatures either unpreserved or preserved with one of ten different stabilizers. Additionally, microbial profiles of aliquots taken before and after homogenization were compared. Feces were collected from 15 healthy volunteers (11 women and 4 men; 20-46 years of age). Ten specimens were collected in the laboratory, while the remaining 5 were collected offsite and returned to the laboratory within 3 h of collection.  Each specimen was sampled and then a portion was homogenized and aliquoted to test the effects associated with different stabilizers. Matched aliquots were immediately frozen at -20°C (control, homogenized and not homogenized), mixed with RNAlater, Tris-EDTA, 95% ethanol, PrimeStore MTM, Stratec, OMNIgene-Gut, Norgen, DNA/RNA Shield, Fecal Swab, or water and placed on a Whatman FTA card. Specimens were stored  14 days total with fixed temperature variation (3 days at room temperature, 3 days at 4°C, 3 days at room temperature, 3 days at 40°C and 2 days at room temperature). DNA was extracted from fecal specimens using the NucleoSpin DNA Stool Kit and was quantified by a NanoDrop spectrophotometer. A 16S metagenomic sequencing library was constructed using the Nextera XT Index Kit and sequenced on a MiSeq instrument. Samples with <30,000 reads were discarded, and reads were processed using QIIME 2; chimeras were removed with DADA2. Taxonomic assignment was performed using Kraken.

   Summary of Findings:

   Comparison of homogenized and non-homogenized frozen aliquots of feces specimens showed that homogenization significantly reduced inter-aliquot variability (P=0.002). DNA yield was greater from fecal specimens stored without stabilizer (mixed with water) than from any of the stabilized specimens. DNA yield was dependent on the preservative used; DNA recovery was the lowest from fecal specimens that were preserved with Stratec, DNA/RNA Shield or on FTA cards, and the highest DNA recovery was from fecal specimens that were frozen directly (unpreserved). DNA purity was also low (A260/280 nm <1.7) when DNA was isolated from specimens preserved with Stratec, DNA/RNA Shield or on FTA cards. While the alpha diversity (observed richness and Shannon index) was similar among the majority of specimens, regardless of preservation method, they were lower for fecal specimens preserved with Stratec. Specimens preserved with Norgen, DNA/RNA Shield, OMNIgene-Gut and PrimeStore MTM had microbial profiles that were most similar (lowest Bray Curtis, Jaccard, and Aitchison distances) to the immediately frozen control specimens, with the largest differences found for Stratec-preserved specimens followed by specimens preserved on FTA cards and Tris EDTA-preserved specimens. Interestingly, the differences in microbial profiles that were observed between case-matched fecal specimens that were immediately frozen and those that were either unpreserved or preserved with Tris EDTA or on FTA cards were similar, although the magnitude of the differences between these case-matched aliquots was smaller than observed between specimens from different individuals..  Stabilizer-specific differences in microbial profiles were found at the phyla and genera level. Among the abundant phyla, Actinobacteria and Proteobacteria tended to be overestimated and Firmicutes and Bacteroidetes underestimated in preserved specimens relative to frozen controls, but in Norgen-preserved specimens only Lentisphaerae were significantly affected. Importantly, in unpreserved specimens, there was a decrease in Firmicutes and an increase in Proteobacteria, Actinobacteria and Lentisphaerae relative to frozen controls. At the genus level, alterations in Tris EDTA and FTA card stabilized specimens were influenced by oxygen status (P=0.025 and P=0.029, respectively), and alterations in DNA/RNA Shield-, PrimeStore MTM- and Stratec-stabilized specimens were influenced by Gram stain status (P=0.002, P=0.0027 and P=0.002, respectively).

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	DNA/RNA Shield Primestore MTM EDTA FTA Card Frozen Stratec OMNIgene GUT Norgen stool preservation agent Ethanol
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Specimen sub-sampling

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