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Reliability of a participant-friendly fecal collection method for microbiome analyses: a step towards large sample size investigation.

Author(s): Szopinska JW, Gresse R, van der Marel S, Boekhorst J, Lukovac S, van Swam I, Franke B, Timmerman H, Belzer C, Arias Vasquez A

Publication: BMC Microbiol, 2018, Vol. 18, Page 110

PubMed ID: 30189859 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared DNA concentration and purity, microbial profiles, alpha diversity, and bacterial relative abundance in matched fresh fecal specimens stored at 4°C to those collected using the OMNIgene-GUT Kit and stored at room temperature for up to 24 h or 7 days and when DNA was isolated by two different extraction methods. Fecal specimens were collected from 14 healthy participants (7 males, 7 females) and immediately stored at 4°C or collected using the OMNIgene-GUT Kit and stored at room temperature ≤24 h or 7 days. DNA was extracted from all specimens using the QIAamp DNA Mini Kit. To compare the effects of extraction methods, specimens collected using the OMNIgene-GUT Kit and stored at room temperature ≤24 h were also extracted using the PowerFecal DNA Isolation Kit. The bacterial 16S rRNA V3-V4 regions were amplified using a 2-step PCR method and amplicon concentration and 260/230 ratios were assessed by NanoDrop. Sequencing was performed on a MiSeq platform and 16S rRNA gene copy numbers were determined by real-time PCR. Alpha diversity was assessed by operational taxonomic units (OTUs) counts and Shannon diversity index. Beta diversity was assessed by principal component analysis.

   Summary of Findings:

   Mean DNA concentration was lower in specimens stored in OMNIgene-GUT kits at room temperature for 7 days than those stored ≤24 h (223.27 ng/uL versus 267.22 ng/uL) and compared to those stored at 4°C (304.60 ng/uL) but the differences were not statistically significant (P>0.05, all). Similarly, absorbance at 260/230 ratios were comparable between fresh and OMNIgene-GUT specimens stored at room temperature ≤24 h or 7 days (1.67 ± 0.20, 1.66 ± 0.16, and 1.60 ± 0.19; respectively, P>0.05 for all). Mean DNA concentration and purity were significantly lower when DNA was extracted from OMNIgene-GUT specimens with the PowerFecal DNA Isolation Kit compared to QIAamp (41.31 ng/uL versus 267.22 ng/uL, P<0.001 and 1.02 versus 1.66, P<0.001; respectively).

   There were 3,532,503 total reads associated to 78,470 OTUs and 49,388 observed bacterial species across all specimens. Lower alpha diversity scores were found between OMNIgene-GUT specimens when DNA was extracted using the PowerFecal DNA Isolation Kit compared to QIAamp (P>0.001) but no differences were observed between storage methods. While phylum relative abundances were not significantly different between OMNIgene-GUT specimens stored at room temperature ≤24 h and 7 days, relative abundances of Bacteroidetes, Actinobacteria, and Cyanobacteria were significantly different between fresh specimens stored at 4°C and OMNIgene-GUT specimens stored at room temperature ≤24 h (P<0.05, all) although only differences in Cyanobacteria abundance were statistically significant when OMNIgene-GUT specimens were stored for 7 days (P=0.045). OMNIgene-GUT specimens showed significantly higher abundance of Bacteroidetes, Cyanobacteria, and Proteobacteria (P<0.01, P<0.05, and P<0.01; respectively) and lower abundance of Firmicutes and Actinobacteria (P<0.01, both) when DNA was extracted using the PowerFecal DNA Isolation Kit compared to extraction with QIAamp. Principal Component Analysis revealed that specimens for 12 of 14 participants strongly clustered intra-individually, regardless of storage method, but were only loosely clustered by extraction method. MiSeq results were confirmed by real-time PCR with no significant differences in bacterial abundances among the storage conditions, but significantly lower amounts of Bacteroides, Bifidobacterium, and Clostridium OMNIgene-GUT specimens when extraction was with the PowerFecal DNA Isolation Kit compared to QIAamp (P<0.001, all).

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	PCR
   DNA	Next generation sequencing
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage conditions	Fresh at 4°C OMNIgene-GUT Kit at room temperature
   Storage	Time at room temperature	≤24 h 7 days
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA Mini Kit PowerFecal DNA Isolation Kit
   Real-time qPCR Specific	Targeted nucleic acid	Bacterial 16S rRNA gene V4 region
   Next generation sequencing Specific	Targeted nucleic acid	Bacterial 16S rRNA gene V4 region

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