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Fecal sample collection methods and time of day impact microbiome composition and short chain fatty acid concentrations.

Author(s): Jones J, Reinke SN, Ali A, Palmer DJ, Christophersen CT

Publication: Sci Rep, 2021, Vol. 11, Page 13964

PubMed ID: 34234185 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated variability in the bacteriome and mycobiome that occurred between subjects, between bowel movements (first morning versus the next within 25 h) and within a specimen using fecal specimens from six healthy women and V4 16S rRNA and ITS2 sequencing, respectively.  Additionally, the bacteriome and mycobiome were compared among aliquots from the same bowel movement that were frozen or stored for 12 days at room temperature in Norgen or OMNIgene-Gut tubes. Three fecal specimens were self-collected at home by six healthy volunteers (25-40 years), with the first two collected in the morning and the third at the next bowel movement after the second (<25 h). At the first collection, three aliquots and the remaining specimen (whole specimen) were frozen for transport (details not provided), and on arrival at the lab stored at -80°C, thawed at 4°C, homogenized, aliquoted for sequencing and refrozen at either -80°C or -20°C (there is a discrepancy between the temperature noted in the text and the figure). At the second and third collection, specimens were frozen and transported to the laboratory where aliquots were placed in OMNIgene-Gut and Norgen Stool Nucleic Acid Collection and Preservation Tubes and stored at room temperature for 12 days while the remainder of the fecal specimen was homogenized and stored at either -80°C or -20°C (there is a discrepancy between the temperature noted in the text and the figure). DNA was extracted from frozen specimens (after they were thawed on ice) and preserved specimens (after shaking) using the QIAamp Power Fecal DNA Kit on a QIAcube. The V4 region of 16S rRNA (bacterial) and ITS2 (Fungal) DNA was PCR amplified with barcoded primers. Sequencing libraries were prepared from equimolar concentrations of the bacterial and fungal libraries and pair-end sequenced using an Illumina MiSeq instrument and the V2 500 cycle Kit.  Reads were demultiplexed, quality filtered, trimmed and those with ambiguous bases or >2 errors were discarded with no mismatches allowed. Amplicon sequence variants (ASVs) were inferred using the pseudo-pooled method and those meeting overlap and mismatch thresholds (≥60 bp and ≤ 1 mismatch for 16S V4 and ≥30 bp and no mismatches for ITS2) were retained at ≥251 bp (16S V4) or ≥150 bp (ITS2) and classified using the “Classification for 16S sequence variants the Genome Taxonomy” reference database (release 95) for 16SV4 and the UNITE general FASTA release for Fungi (version 18.11.2018). α-diversity was analyzed using R and β-diversity was compared using PERMANOVA in PRIMER-e v7 and visualized using principal coordinate analysis (PCoA).

   Summary of Findings:

   Principal coordinate analysis (PCoA) based on the bacteriome clearly separated specimens based on subject, but the clustering based on the mycobiome was less distinct; nevertheless,β-diversity (Euclidian and Bray–Curtis distances) showed significant differences in the bacterial and fungal community between subjects (PERMANOVA, P<0.02). Hierarchical clustering based on the mycobiome or bacteriome clustered specimens based on subject, as the three individual aliquots, the combined aliquot and the whole (remaining) fecal specimen grouped together, with more distinction between subjects in the bacteriome clustering than the mycobiome clustering. While phylogenetic diversity (3 of 3 subjects) was as variable within a fecal specimen as between three different bowel movements, the variability among bowel movements was greater than the variability within a single specimen for Shannon diversity (5 of 6 subjects), and the variability in Chao1 species richness within a specimen was greater than the variability between bowel movements (four of six subjects). The second specimen collected in a 25 h period had a non-significant trend toward lower bacterial richness (p = 0.45) and diversity estimates (p = 0.95) compared to the first specimen from 4 of 6 subjects, but no difference in mycobiome diversity between the two specimens was observed. ASV analysis found enrichment of Acidaminococcaceae and Lachnospiraceae and reduced Dialisteraceae and Muribaculac in the second fecal specimen relative to the first.  PCoA based on bacteriome β-diversity (Euclidian and Bray–Curtis distances) primarily showed separation based on subject but some sub-clustering of frozen specimens from those preserved/stored in either the Norgen or OMNIgene-Gut tubes was observed. PERMANOVA confirmed significant differences in both β-diversity metrics between immediately frozen specimens and those preserved with either the Norgen or OMNIgene-Gut tubes (P<0.01), as well as significant differences between those preserved/stored in Norgen versus OMNIgene-Gut tubes (P<0.01 for Bray–Curtis and P=0.04 for Euclidian distance). Regardless of preservation/storage method, Bacteroidaceae was the most abundant family (38% in frozen, 43% in Norgen, and 39% in OMNIgene-Gut), but specimens stored in Norgen or OMNIgene-Gut tubes had significantly more Ruminococcaceae than frozen specimens (17% and 33%, respectively versus 7%, P<0.001).  While the abundance of several phyla were found to differ between frozen specimens and those preserved/stored in Norgen or OMNIgene-Gut tubes, only the lower abundance of Coriobacteria and Negativicutes in frozen relative to OMNIgene-GUT specimens were significant after FDR correction.

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Next generation sequencing Specific	Targeted nucleic acid	V4 16S rRNA ITS2
   Biospecimen Acquisition	Time of biospecimen collection	First bowel movement of the day Next bowel movement (<25 h after first)
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Multiple specimens analyzed Specimen sub-sampling
   Biospecimen Preservation	Type of fixation/preservation	Frozen Norgen stool preservation agent OMNIgene GUT
   Storage	Storage conditions	Room temperature in OMNIgene-Gut tube for 12 d Frozen Room temperature in Norgen Stool tube for 12 d

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2. Study Purpose

   This study investigated the variability in the short chain fatty acid (SCFA) profile that occurred between subjects, between bowel movements (first morning versus next within 25 h) and within a specimen. Additionally, the SCFA profile was compared among fecal aliquots that were frozen or stored for 12 days at room temperature in Norgen or OMNIgene-Gut tubes. Three fecal specimens were self-collected at home by six healthy volunteers (25-40 years) with the first two collected in the morning and the third collected at the next bowel movement after the second specimen (<25 h). At the first collection, three aliquots and the remaining specimen (whole specimen) were frozen for transport (details not provided), and stored at -80°C on arrival at the lab, thawed at 4°C, homogenized, aliquoted for SCFA and refrozen at -80°C or -20°C (there is a discrepancy between the temperature noted in the text and the figure). At the second and third collection, specimens were frozen and transported to the laboratory where aliquots were placed in OMNIgene-Gut and Norgen Stool Nucleic Acid Collection and Preservation Tubes and stored at room temperature for 12 days while the remainder of the fecal specimen was homogenized and stored at -80°C or -20°C (there is a discrepancy between the temperature noted in the text and the figure). SCFA levels were quantified by gas chromatography mass spectrometry (GC–MS).

   Summary of Findings:

   SCFA levels were unaffected by preservation/storage method, but acetic acid levels were highly variable in all subjects. While acetic acid and valeric acid levels were as variable within a fecal specimen as between the three different bowel movements from an individual, the variability among bowel movements was greater than the variability within a single specimen for propionic acid. When consecutive bowel movements were compared the first had lower total SCFA concentration (p = 0.04) and acetic acid concentration (p = 0.03). In Principal component analysis based on SCFE concentration, specimens clustered based on the Bristol Stool Form Scale (BSFS).

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Small molecule	GC-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Time of biospecimen collection	First bowel movement of the day Next bowel movement (<25 h after first)
   Storage	Storage conditions	Room temperature in OMNIgene-Gut tube for 12 d Room temperature in Norgen Stool tube for 12 d Frozen
   Biospecimen Preservation	Type of fixation/preservation	Frozen Norgen stool preservation agent OMNIgene GUT
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Multiple specimens analyzed Specimen sub-sampling

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