scholarly journals Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10837
Author(s):  
Stefan Gavriliuc ◽  
Mason R. Stothart ◽  
Astrid Henry ◽  
Jocelyn Poissant

The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temperature is conventionally thought to be among the most important factors for ensuring reproducibility across marker gene studies, but to date much of the research on this topic has focused on short-term storage in the context of clinical applications. Consequently, it has remained unclear if storage at −80 °C, widely viewed as the gold standard for long-term archival of feces, is truly required for maintaining sample integrity in amplicon-based studies. A better understanding of the impacts of long-term storage conditions is important given the substantial cost and limited availability of ultra-low temperature freezers. To this end, we compared bacterial microbiome profiles inferred from 16S V3–V4 amplicon sequencing for paired fecal samples obtained from a feral horse population from Sable Island, Nova Scotia, Canada, stored at either −80 °C or −20 °C for 4 years. We found that storage temperature did not significantly affect alpha diversity measures, including amplicon sequence variant (ASV) richness and evenness, and abundance of rare sequence variants, nor presence/absence, relative abundances and phylogenetic diversity weighted measures of beta diversity. These results indicate that storage of equine feces at −20 °C for periods ranging from a few months to a few years is equivalent to storage at −80 °C for amplicon-based microbiome studies, adding to accumulating evidence indicating that standard domestic freezers are both economical and effective for microbiome research.

Metabolites ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 93 ◽  
Author(s):  
Cora McHugh ◽  
Thomas Flott ◽  
Casey Schooff ◽  
Zyad Smiley ◽  
Michael Puskarich ◽  
...  

Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at −80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.


2020 ◽  
Vol 100 (10) ◽  
pp. 1345-1355 ◽  
Author(s):  
Stefaniya Boneva ◽  
Anja Schlecht ◽  
Daniel Böhringer ◽  
Hans Mittelviefhaus ◽  
Thomas Reinhard ◽  
...  

Abstract This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.


2016 ◽  
Vol 688 ◽  
pp. 10-16
Author(s):  
Blažej Seman ◽  
Anton Geffert ◽  
Jarmila Geffertova

Wood is loosely stored to ensure continuous production inside paper mills where it is exposed to the effect of external factors. The impact of storage leads to some changes of mechanical and physical properties of wood, but these changes are not the same in all specimens. In this paper, it has been observed that the long term storage of wood influences the impact strength in bending and the permeability of wood for fluids. During the storage, there was a decrease of impact strength in bending of poplar heartwood by 28.3% and oak by 22.1% and mature beech wood by 37.3%. Also, there was decreased a permeability of wood, poplar sapwood 18.3 % and heartwood of 53.9%; oak sapwood by 20.0% and heartwood by 20.3%; beech sapwood 45.8% and mature wood by 48.2%. By decrease of the observed properties of the stored wood, a deterioration a quality of produced pulp can be expected (a higher Kappa number, amount reject and decrease the mechanical properties of pulp).


2021 ◽  
Vol 14 (1) ◽  
pp. 96-105
Author(s):  
V. V. Suskin ◽  
◽  
I. V. Kapyrin ◽  
F. V. Grigorev ◽  
◽  
...  

The article evaluates the impact of a “buried wall” barrier on the long-term safety during the long-term storage1 or in-situ disposal of nuclear legacy facilities, in particular, industrial reservoirs, as well as during the development of near-surface disposal facilities for radioactive waste (RWDF). For assessment purposes, filtration and mass transfer processes have been numerically modelled in the GeRa code based on a case study of a reference near-surface facility. The study explores in which way the available covering screen affects the dynamics of contaminant spread. It evaluates the sensitivity of the results to the dispersion parameter commonly characterized by a high degree of uncertainty.


Author(s):  
Narendra K. Gupta

Drum type packages are routinely used to transport radioactive material (RAM) in the U.S. Department of Energy (DOE) complex. These packages are designed to meet the federal regulations described in 10 CFR 71. In recent years, there has been a greater need to use these packagings to store the excess fissile material, especially plutonium for long term storage. While the design requirements for safe transportation of these packagings are well defined, the requirements for safe long term storage are not well established. Since the RAM contents in the packagings produce decay heat, it is important that they are stored carefully to prevent overheating of the containment vessel (CV) seals to prevent any leakage and the impact limiter to maintain the package structural integrity. This paper analyzes different storage arrays for a typical 9977 packaging for thermal considerations and makes recommendations for their safe storage under normal operating conditions.


2014 ◽  
Vol 670-671 ◽  
pp. 376-381 ◽  
Author(s):  
Aleksey Adamtsevich ◽  
Aleksey Eremin ◽  
Andrey Pustovgar ◽  
Stanislav Pashkevich ◽  
Sergey Nefedov

This article is a stage of the author’s research into the impact of different factors on the hydration of mineral binders. The problem of cement activity decrease due to adsorption moisture influence during long-term storage in normal conditions (20°C and 50% RH) was examined. The influence of storage period in airtight and non-airtight conditions on the kinetics of heat evolution during hydration as well as on phase composition of Portland cement was characterized using experimental methods.


2022 ◽  
Vol 31 ◽  
pp. 100770
Author(s):  
Lucas Mallmann Wendt ◽  
Vagner Ludwig ◽  
Fabiane Portella Rossato ◽  
Magno Roberto Pasquetti Berghetti ◽  
Erani Eliseu Schultz ◽  
...  

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