Changes in Microbiota Profiles After Prolonged Frozen Storage of Stool Suspensions.
Author(s): Dorsaz S, Charretier Y, Girard M, Gaïa N, Leo S, Schrenzel J, Harbarth S, Huttner B, Lazarevic V
Publication: Front Cell Infect Microbiol, 2020, Vol. 10, Page 77
PubMed ID: 32185143 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of frozen storage duration of fecal specimen suspensions and DNA extraction method on DNA yield and on alpha diversity. DNA yield was determined fluorometrically and by real-time PCR and alpha diversity was investigated by next-generation sequencing (NGS) of the bacterial 16S rRNA gene.
Conclusion of Paper
DNA yields were higher in fecal specimens extracted using the NS-MAG method than the MOLZ method. DNA yields decreased with increased storage time, regardless of extraction method. Bacterial alpha diversity, absolute abundance of all bacterial taxa, and relative abundance of Bacteroidetes were lower in fresh specimens extracted using the MOLZ method compared to NS-MAG method and demonstrated storage duration-dependent decreases. Further, principal coordinates analysis (PCoA) of operational taxonomic unit (OTU) relative abundances revealed clustering based on DNA extraction method which were unchanged with storage duration in NS-MAG-extracted specimens but dissimilarities were notably increased in MOLZ-extracted specimens.
Studies
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Study Purpose
This study investigated the effects of frozen storage duration of fecal specimen suspensions and DNA extraction method on DNA yield as measured fluorimetrically and by real-time qPCR and on alpha diversity evaluated by NGS of the bacterial 16S rRNA gene. Specimens collected from a single healthy volunteer were suspended in a NaCl solution using a commercial blender and sequentially sieved using 1, 0.5, and 0.25-mm Nylon filters. The suspension was centrifuged at 2,800 × g for 20 min, supernatant was discarded, and cold 80% glycerol in NaCl was added at one tenth of the volume of the initial NaCl stool suspension. DNA was extracted from aliquots immediately (fresh) or after frozen storage at -80°C for 1 week or 1, 3, 6, 9, 12, or 18 months. DNA was extracted using an Ultra-Deep Microbiome Prep Kit for selective enrichment of bacterial/fungal DNA following manufacturer’s instructions (MOLZ method) or using a protocol with no bacterial/fungal DNA enrichment (NS-MAG method). The NS-MAG method consisted of a 20-min bead-beating mechanical disruption in a Nucleospin Bead Tube, chemical lysis and DNA purification with a MagCore Genomic DNA Tissue Kit, and DNA elution with Tris–HCl. DNA was quantified using a fluorometer and by real-time qPCR amplification of the V3 region of bacterial 16S rRNA genes. Sequencing libraries were generated using the KAPA2G Robust HotStart ReadyMix and the library was sequenced on an Illumina MiSeq platform.
Summary of Findings:
DNA yields from fecal specimens were higher after extraction using the NS-MAG method than the MOLZ method and decreased with increased storage time, regardless of extraction method. Bacterial alpha diversity as determined by operational taxonomic units (OTUs) and the Shannon diversity index was higher when DNA was extracted using NS-MAG than MOLZ method. All of the 81 OTUs with an average relative abundance >0.1% in fresh specimens were identified after DNA extraction using either method but 27 OTUs exhibited a 5-fold decrease in relative abundance in MOLZ-extracted specimens compared to NS-MAG specimens. Relative abundance of Bacteroidetes was lower in fresh specimens extracted using the MOLZ method compared to NS-MAG (2.5% versus 21.7%) and decreased with storage duration, decreasing to an average of 0.08% after 18 months. Absolute abundance of all bacterial taxa were lower when DNA was extracted using the MOLZ method compared to the NS-MAG method and were not affected by frozen storage duration except Firmicutes abundances which decreased slightly with frozen storage duration in MOLZ-extracted specimens. Principal coordinates analysis (PCoA) of OTU relative abundances revealed specimen clustering based on DNA extraction method with NS-MAG-extracted specimens clustering more tightly than MOLZ-extracted specimens. Dissimilarities in MOLZ specimens increased only slightly with frozen storage up to 12 months and then rose markedly at months 15 and 18. In contrast, dissimilarity in NS-MAG specimens was unchanged with storage duration.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA Next generation sequencing DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 week
1 month
3 months
6 months
9 months
12 months
18 months
Real-time qPCR Specific Targeted nucleic acid V3 region of bacterial 16S rRNA genes
Next generation sequencing Specific Targeted nucleic acid V3 region of bacterial 16S rRNA genes
Analyte Extraction and Purification Analyte isolation method MOLZ method
NS-MAG method