Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Method optimization for fecal sample collection and fecal DNA extraction.

Author(s): Mathay C, Hamot G, Henry E, Georges L, Bellora C, Lebrun L, de Witt B, Ammerlaan W, Buschart A, Wilmes P, Betsou F

Publication: Biopreserv Biobank, 2015, Vol. 13, Page 79-93

PubMed ID: 25880472 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared microbial profiles in fecal specimens stored at room temperature or frozen in different stabilization solutions to those snap-frozen immediately after collection. Effects of storage duration on relative abundance before and after freezing were also investigated. Specimens were collected from two healthy volunteers into a sterile container, mixed with phosphate-buffered saline, homogenized by rigorous mixing using a sterile spatula, aliquoted, and snap-frozen at -80°C in a sterile feces container within 2 h of collection or placed in an OMNIgene-GUT Kit, in RNAlater in a ParasiTrap Tube, in AquaStool in a ParasiTrap Tube, in RNAssist in a ParasiTrap Tube, in a DNA Genotek’s CP-150 Tube, in PerkinElmer/Chemagen SEB lysis buffer in a ParasiTrap Tube, or in a sterile feces container without a stabilizing solution and then stored at room temperature for 24 h. To assess homogeneity, relative abundance of dominant genera were compared in frozen cores taken using the CryoXtract CXT353 System in specimens from two donors that were immediately frozen at -80°C in a sterile feces container (non-homogenized) or aliquoted into an OMNIgene-GUT Kit, in RNAlater in a ParasiTrap Tube, or in AquaStool in a ParasiTrap Tube; homogenized using either using the metallic bead in the OMNIgene-GUT tubes or by the cap press in the ParasiTrap tubes; transferred into 2 mL cryovials, and stored frozen at -80°C for at least 24 h. To examine effects of pre-freezing delay, specimens from two donors were collected and aliquoted into OMNIgene-GUT Tubes and frozen immediately at -80°C or after 24 h, 1 week, or 2 weeks at room temperature. To examine effects of frozen storage duration, a specimen from a single donor was aliquoted into OMNIgene-GUT Tubes and then frozen at -80°C for 24 h or 2 years. DNA was extracted using the DNA Blood 4k Kit special H24. Total DNA quantification and A260/A280 ratio were determined by spectrophotometry and double-stranded DNA was quantified by fluorometry. The bacterial 16S rRNA gene V4 region was amplified by PCR, sequenced on a MiSeq platform, and clustered into operational taxonomic units (OTUs). Alpha diversity was assessed by OTU richness, Shannon–Wiener index, and Simpson’s evenness. Beta diversity was determined using the Bray–Curtis dissimilarity index.

   Summary of Findings:

   Total DNA yields varied among the preservation conditions but were higher from RNAlater, OMNIgene-GUT, and AquaStool specimens and lower from non-stabilized and PE Lysis buffer-preserved specimens stored at room temperature compared to matched snap-frozen specimens. Double-stranded DNA yields were higher for all preservation conditions compared to the snap-frozen specimens with the exception of those preserved in RNAssist. Mean A260/A280 ratios ranged between 1.7 and 1.9 for all preservation conditions with the exception of specimens preserved in PE Lysis buffer (mean ratio=1.55). There were no significant differences in OTU richness, alpha diversity, evenness, or beta diversity between preservation conditions (P>0.05, all). Both total and double-stranded DNA yields were higher in the frozen cores obtained from the homogenized specimens frozen in stabilization solutions (AquaStool, RNAlater, and OMNIgene-GUT) compared to the snap-frozen cores. The proportion of the dominant genera did not vary significantly between the cores extracted from the homogenized specimens stored in stabilization solutions (ranges from 1.2 – 17.8), but the non-homogenized snap-frozen specimens showed a non-significant higher CV% (< 30%) between cores for all dominant genera (ranges from 5.4 – 26.5). For specimens stored in OMNIgene-GUT Tubes and subjected to a delay before frozen storage, OTU richness, alpha diversity, beta diversity, and evenness were comparable between the different time points (P>0.05, all) and no significant variation (CV% < 30%) in the proportion of the dominant genera was observed. The relative abundances of genera (OTU >1%) was not significantly different between specimens in OMNIgene-GUT Tubes stored at -80°C for 24 h and those stored for 2 years.

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Next generation sequencing
   DNA	Spectrophotometry
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh) OMNIgene GUT RNAlater AquaStool RNAssist PerkinElmer/Chemagen SEB lysis buffer DNA Genotek’s CP-150
   Storage	Storage temperature	Room temperature -80°C
   Storage	Storage duration	24 h 2 years
   Storage	Time at room temperature	24 h 1 week 2 weeks
   Next generation sequencing Specific	Targeted nucleic acid	Bacterial 16S rRNA gene V4 region
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Multiple specimens analyzed
   Biospecimen Aliquots and Components	Biospecimen mixing	Homogenized Not homogenized

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