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An alternative storage method for characterization of the intestinal microbiota through next generation sequencing.

Author(s): Ribeiro RM, Souza-Basqueira M, Oliveira LC, Salles FC, Pereira NB, Sabino EC

Publication: Rev Inst Med Trop Sao Paulo, 2018, Vol. 60, Page e77

PubMed ID: 30517247 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of preservation method and storage temperature of fecal specimens on alpha and beta diversity evaluated by NGS of the bacterial 16S rRNA gene. Specimens were collected from 10 healthy participants between the ages of 23 and 49 (mean age: 36 years) using the Fisherbrand Commode Specimen Collection System and divided into five 20 g aliquots within 1 h of collection. One aliquot was extracted immediately after collection (fresh), two aliquots were subjected to freezing at -20°C or -80 °C for 48 h, and the other two aliquots were stored in a guanidine HCl/EDTA solution at room temperature or 4 °C for 48 h. DNA was extracted using a Power Soil DNA Isolation Kit. The V4 hypervariable regions of bacterial and archeal 16S rRNA gene were amplified by PCR and sequenced using an Ion Torrent PGM platform.

   Summary of Findings:

   The number of NGS reads, alpha diversity, and beta diversity were not significantly different between fresh specimens, specimens frozen at -20°C or -80°C, and specimens stored in guanidine at 4°C or room temperature. Variations in relative abundance of operational taxonomic units (OTUs) were due to inter-individual variability and did not cluster based on storage condition. Higher levels of Firmicutes were noted in specimens stored for 48 h at -20°C and -80°C compared to fresh samples but differences were not significant.

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh) Guanidine hydrochloride
   Next generation sequencing Specific	Targeted nucleic acid	V4 variable region of the 16S rRNA gene
   Storage	Storage temperature	4°C -20°C 80°C Room temperature

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