Comparison of DNA stabilizers and storage conditions on preserving fecal microbiota profiles.
Author(s): Chen CC, Wu WK, Chang CM, Panyod S, Lu TP, Liou JM, Fang YJ, Chuang EY, Wu MS
Publication: J Formos Med Assoc, 2020, Vol. 119, Page 1791-1798
PubMed ID: 32111519 PubMed Review Paper? No
Purpose of Paper
This paper compared the fecal microbiomes of samples collected from two different locations of the same fecal specimen that were frozen immediately at -80°C or stored in Stratec or OMNIgene.GUT at room temperature for 0, 3, or 7 days and then frozen; fecal specimens were collected from healthy men.
Conclusion of Paper
In total, 128,038 high-quality reads (5,627-82,041 reads per sample) mapped to 32,355 operational taxonomic unit (OTUs). Compared to matched specimens that were frozen immediately without preservation, fecal samples that were preserved in OMNIgene.GUT or Stratec showed changes in the relative abundance of the high abundance genera Faecalibacteria, Suterella, Lachnospira, Fusobacteria, Prevotella, and Ruminococcus as well as the disease-associated genera Bifidobacteria, Faecalibacteria, Fusobacteria, Prevotella, and Roseburia, but the changes were dependent on the subject and storage duration. The relative abundance of the phyla differed between specimens, with a significant difference in Shannon Index found among individuals, but not preservation methods, storage durations, or sampling location within the fecal specimen. The relative abundance of each OTU was strongly correlated between specimens that were frozen immediately and those that were frozen after 3 or 7 days in either solution. The largest Bray-Curtis and Jaccard distances were inter-subject, followed by between timepoints, with the smallest differences found between the fecal sampling locations. Principal coordinates analysis (PCoA) based on Bray-Curtis distances clustered specimens by subject (accounting for 60.9% of the variability), but only minimally by preservation method (accounting for 5.2% of the variability) or storage duration at room temperature (accounting for 4.4% of the variability). Further analysis found that differences between Stratec-preserved specimens and unpreserved specimens were caused by a shift in the abundance of Faecalibacteria, but no specific taxa were identified in OMNIgene.GUT specimens.
Studies
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Study Purpose
This study compared the fecal microbiome of matched specimens that were collected from two locations within the same fecal specimen and stored at -80°C or in Stratec or OMNIgene.GUT at room temperature for 0, 3 or 7 days; fecal specimens were collected from healthy men. Fecal specimens were collected from four healthy men (age 31-40 years). Immediately after defecation, matched aliquots were obtained from each end of the fecal specimen and immediately frozen at -80°C or placed in a Stratec Stool collection tube (3 aliquots) or OMNIgene.GUT Kit (3 aliquots). The specimens collected in Stratec Stool collection tubes and OMNIgene.GUT were frozen at -80°C after 0 h, 3 days, and 7 days at ambient temperature. DNA was extracted from fecal specimens with the QIAmp Fast DNA Stool Mini Kit and quantified by spectrophotometer. 16S sequencing libraries were prepared using the protocol in the Illumina 16S sample preparation guide and pair-end sequenced on an Illumina Miseq instrument using v3 reagents. Raw reads were filtered and merged using Paired-End Read Merger, analyzed using QIIME v1.9.1, and operational taxonomy units (OTUs) were identified using the Greengenes database and an identity cut-off of 97%. Shannon and Chao 1 diversity indices were calculated using the R package Vegan.
Summary of Findings:
In total, 128,038 high-quality reads (5,627-82,041 reads per sample) mapped to 32,355 OTUs. Firmicutes and Bacteroidetes were the most common phyla in the majority of specimens, though Verrucomicrobia accounted for >50% of the species detected in one end of one frozen specimen. Compared to specimens frozen immediately without preservation, matched specimens preserved in OMNIgene.GUT or Stratec showed changes in the relative abundance of the high abundance genera Faecalibacteria, Suterella, Lachnospira, Fusobacteria, Prevotella, and Ruminococcus as well as the disease-associated genera Bifidobacteria, Faecalibacteria, Fusobacteria, Prevotella, and Roseburia, but the changes were dependent on subject and storage duration. The relative abundance of the phyla differed between specimens, with a significant difference in Shannon Index found among individuals, but not preservation methods, storage durations, or sampling location within the fecal specimen. The relative abundance of each OTU was strongly correlated between specimens frozen immediately and those frozen after 3 or 7 days in Stratec (R=0.93 and R=0.89, respectively) or OMNIgene.GUT (0.93 and R=0.83, respectively). The largest Bray-Curtis and Jaccard distances were inter-subject, followed by between timepoints, with the smallest differences found between fecal sampling locations. PCoA based on Bray-Curtis distances clustered specimens by subject (accounting for 60.9% of the variability), but only minimally by preservation method (accounting for 5.2% of the variability) or storage duration at room temperature (accounting for 4.4% of the variability). Further analysis found that differences between Stratec-preserved specimens and unpreserved specimens were caused by a shift in the abundance of Faecalibacteria, but no specific taxa were identified in OMNIgene.GUT specimens.
Biospecimens
Preservative Types
- Other Preservative
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Biospecimen heterogeneity Multiple specimens analyzed
Storage Time at room temperature 0 h
3 days
7 days
Biospecimen Preservation Type of fixation/preservation OMNIgene GUT
Frozen
Stratec