scholarly journals PSV-36 Optimization of Collection, Storage, and Extraction of Samples for Large-Scale 16s Sequencing Project

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 338-338
Author(s):  
Robert D Jacobs ◽  
Mary Beth E Gordon ◽  
Morghan Bowman
Keyword(s):  
Large Scale ◽  
Storage Conditions ◽  
Primary Objective ◽  
Rna Transport ◽  
Sample Collection ◽  
16S Sequencing ◽  
Sequencing Project ◽  
Horse Owner ◽  
Zymo Research

Abstract Correlations between the microbiome of the horse and various physiological parameters continue to be determined. Most evaluations of the equine microbiome have been regionally focused with limited numbers or variability. In order to elucidate the role of the microbiome on equine health, a large-scale trial has been initiated with a primary objective of analyzing and characterizing the microbiome of the horse. A study of this scope relies on the horse owner to collect samples from various locations and ship them to a centralized lab. Therefore, collection protocols and extraction techniques need to be optimized to maintain sample integrity. A trial was conducted to evaluate the storage conditions and extraction methodologies necessary to develop an optimized method of sample collection, preparation, and extraction. Samples were collected from a single horse and subjected to one of four treatments in duplicate (1-fresh, 2-frozen, 3-stored in 99% EtOH, and 4-stored in a commercially available DNA/RNA transport medium; DNA/RNA Shield®, Zymo Research, Irvine, CA). Frozen samples were placed at -80◦ C immediately following collection and stored for 48-hr. Frozen samples were thawed immediately prior to extraction. All other treatments were stored as dictated by treatment at room temperature for 48-hr prior to extraction to mimic shipping. Samples were processed utilizing a commercially available DNA extraction kit (Quick-DNA™ Fecal/Soil Microbe Miniprep, Zymo Research). DNA extract was analyzed for quantity utilizing a Qubit Fluorometer (ThermoFisher, Waltham, MA), and quality via spectrophotometry (NanoDrop 2000; ThermoFisher, Waltham, MA). Swabs stored in DNA/RNA Shield® yielded higher concentrations of DNA with superior 260/280 values than other treatments. These data indicate that storage of fecal swabs in an appropriate buffer combined with optimized extraction protocols can deliver adequate extracts that can be utilized for 16s sequencing and validates the use of this method for large-scale microbiome analysis.

scholarly journals Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Diagnostics ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 234 ◽  
Author(s):  
Eun Young Lee ◽  
Eun-Ju Lee ◽  
Hana Yoon ◽  
Dong Hyeon Lee ◽  
Kwang Hyun Kim
Keyword(s):  
Cost Efficiency ◽  
High Sensitivity ◽  
Storage Conditions ◽  
Sample Storage ◽  
Sample Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Zymo Research ◽  
Free Circulating Dna ◽  
Commercial Kits

Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

scholarly journals A comprehensive automated pipeline for human microbiome sampling, 16S rRNA gene sequencing and bioinformatics processing

2018 ◽  
Author(s):  
LW Hugerth ◽  
M Seifert ◽  
AAL Pennhag ◽  
J Du ◽  
MC Hamsten ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Large Scale ◽  
Human Microbiome ◽  
Population Level ◽  
16S Rrna Gene Sequencing ◽  
Extraction Methods ◽  
Storage Conditions ◽  
Rrna Gene ◽  
Sample Collection ◽  
Dna Extraction Methods

ABSTRACTThe advent of affordable high-throughput DNA sequencing has opened up a golden age of studies in the human microbiome. In order to understand the role of the human microbiota, standardized methods for large-scale, population-level studies are needed to avoid underpowered or poorly designed studies. The biggest bottlenecks to population-level microbiomics are sample collection, storage and DNA extraction. Here, we describe a flexible automated approach to process intestinal biopsies, fecal samples and vaginal swabs from sample collection to OTU table. We have evaluated storage conditions, DNA extraction methods, PCR strategies and bioinformatic pipelines for these three sample types, and present here a set of guidelines and best practices for each of these steps.

scholarly journals Stability of amino acids and related amines in human serum under different preprocessing and pre-storage conditions based on iTRAQ®-LC-MS/MS

Biology Open ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. bio055020
Author(s):  
Zhuoling An ◽  
Chen Shi ◽  
Pengfei Li ◽  
Lihong Liu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Serum ◽  
Large Scale ◽  
Storage Conditions ◽  
Sample Collection ◽  
Serum Samples ◽  
Treatment Measures ◽  
Isobaric Tagging ◽  
Pre Treatment

ABSTRACTAmino acid analysis or metabonomics requires large-scale sample collection, which makes sample storage a critical consideration. However, functional amino acids are often neglected in metabolite stability studies because of the difficulty in detecting and accurately quantifying them with most analysis methods. Here, we investigated the stability of amino acids and related amines in human serum following different preprocessing and pre-storage procedures. Serum samples were collected and subjected to three storage conditions; cold storage (4°C), room temperature storage (22°C), and freezing (−80°C). The concentration of amino acids and related amines were quantified using iTRAQ®-LC-MS/MS with isobaric tagging reagents. Approximately 54.84%, 58.06%, and 48.39% of detectable and target analytes were altered at the 4°C condition, 22°C condition, and when subjected to freeze-thaw cycles, respectively. Some amino acids which are unstable and relatively stable were found. Our study provides detailed amino acid profiles in human serum and suggests pre-treatment measures that could be taken to improve stability.

scholarly journals Collection of Human Genomic DNA From Buccal Cells for Genetics Studies: Comparison Between Cytobrush, Mouthwash, and Treated Card

2005 ◽  
Vol 2005 (3) ◽  
pp. 291-296 ◽  
Author(s):  
Claire Mulot ◽  
Isabelle Stücker ◽  
Jacqueline Clavel ◽  
Philippe Beaune ◽  
Marie-Anne Loriot
Keyword(s):  
Large Scale ◽  
Storage Conditions ◽  
Sample Collection ◽  
Buccal Cells ◽  
Genetic Studies ◽  
Dna Yield ◽  
Dna Collection ◽  
Alternative Sources ◽  
And Storage

Alternative sources such as buccal cells have already been tested for genetic studies and epidemiological investigations. Thirty-seven volunteers participated in this study to compare cytology brushes, mouthwash, and treated cards for DNA collection. Quantity and quality of DNA and cost and feasibility were assessed. The mean DNA yield at 260 nm was found to be3.5,4, and2.6μg for cytobrushes, mouthwashes, and treated cards, respectively. A second quantification technique by fluorescence showed differences in the DNA yield with1.1and5.2μg for cytobrushes and mouthwash, respectively. All buccal samples allowed isolation of DNA suitable for polymerase chain reaction. According to the procedure of sample collection, the yield and purity of collected DNA, and storage conditions, the use of cytobrush appears to be the more appropriate method for DNA collection. This protocol has been validated and is currently applied in three large-scale multicentric studies including adults or children.

The Role of Sense of Direction and Other Factors During Navigation in a Large-Scale, Unconstrained Environment

2013 ◽  
Author(s):  
Elisabeth J. Ploran ◽  
Ericka Rovira ◽  
James C. Thompson ◽  
Raja Parasuraman
Keyword(s):  
Large Scale ◽  
Sense Of Direction

Magnetic Properties of xCeO2•(50 − x) PbO•50B2O3 Glasses and Glass Ceramics

2017 ◽  
Vol 13 (1) ◽  
pp. 4486-4494 ◽  
Author(s):  
G.El Damrawi ◽  
F. Gharghar
Keyword(s):  
Magnetic Properties ◽  
Large Scale ◽  
Glass Ceramics ◽  
Magnetic Behavior ◽  
Crystal Formation ◽  
Dual Roles ◽  
Effective Agent ◽  
Ordered Structures ◽  
Essential Change

Cerium oxide in borate glasses of composition xCeO2·(50 − x)PbO·50B2O3 plays an important role in changing both microstructure and magnetic behaviors of the system. The structural role of CeO2 as an effective agent for cluster and crystal formation in borate network is clearly evidenced by XRD technique. Both structure and size of well-formed cerium separated clusters have an effective influence on the structural properties. The cluster aggregations are documented to be found in different range ordered structures, intermediate and long range orders are the most structures in which cerium phases are involved. The nano-sized crystallized cerium species in lead borate phase are evidenced to have magnetic behavior.  The criteria of building new specific borate phase enriched with cerium as ferrimagnetism has been found to keep the magnetization in large scale even at extremely high temperature. Treating the glass thermally or exposing it to an effective dose of ionized radiation is evidenced to have an essential change in magnetic properties. Thermal heat treatment for some of investigated materials is observed to play dual roles in the glass matrix. It can not only enhance alignment processes of the magnetic moment but also increases the capacity of the crystallite species in the magnetic phases. On the other hand, reverse processes are remarked under the effect of irradiation. The magnetization was found to be lowered, since several types of the trap centers which are regarded as defective states can be produced by effect of ionized radiation. 

The Impact of Quantitative Easing on Emerging Markets – Literature Review

e-Finanse ◽  
2018 ◽  
Vol 14 (4) ◽  
pp. 67-76
Author(s):  
Piotr Bartkiewicz
Keyword(s):  
Emerging Markets ◽  
Large Scale ◽  
Empirical Literature ◽  
Quantitative Easing ◽  
Methodological Choices ◽  
The Subject ◽  
Methodological Approaches ◽  
The Impact ◽  
Sufficient Precision

AbstractThe article presents the results of the review of the empirical literature regarding the impact of quantitative easing (QE) on emerging markets (EMs). The subject is of interest to policymakers and researchers due to the increasingly larger role of EMs in the world economy and the large-scale capital flows occurring after 2009. The review is conducted in a systematic manner and takes into consideration different methodological choices, samples and measurement issues. The paper puts the summarized results in the context of transmission channels identified in the literature. There are few distinct methodological approaches present in the literature. While there is a consensus regarding the direction of the impact of QE on EMs, its size and durability have not yet been assessed with sufficient precision. In addition, there are clear gaps in the empirical findings, not least related to relative underrepresentation of the CEE region (in particular, Poland).

Age Advantages of Emotional Experience during the COVID-19 Pandemic are Robust, but Diminished Compared to Pre-pandemic Conditions

2020 ◽  
Author(s):  
Rui Sun ◽  
Disa Sauter
Keyword(s):  
Older Adults ◽  
Large Scale ◽  
Negative Emotion ◽  
Emotional Experience ◽  
Negative Emotions ◽  
Older Individuals ◽  
Selection Strategies ◽  
Scale Test ◽  
Prolonged Stress

Getting old is generally seen as unappealing, yet aging confers considerable advantages in several psychological domains (North & Fiske, 2015). In particular, older adults are better off emotionally than younger adults, with aging associated with the so-called “age advantages,” that is, more positive and less negative emotional experiences (Carstensen et al., 2011). Although the age advantages are well established, it is less clear whether they occur under conditions of prolonged stress. In a recent study, Carstensen et al (2020) demonstrated that the age advantages persist during the COVID-19 pandemic, suggesting that older adults are able to utilise cognitive and behavioural strategies to ameliorate even sustained stress. Here, we build on Carstensen and colleagues’ work with two studies. In Study 1, we provide a large-scale test of the robustness of Carstensen and colleagues’ finding that older individuals experience more positive and less negative emotions during the COVID-19 pandemic. We measured positive and negative emotions along with age information in 23,629 participants in 63 countries in April-May 2020. In Study 2, we provide a comparison of the age advantages using representative samples collected before and during the COVID-19 pandemic. We demonstrate that older people experience less negative emotion than younger people during the prolonged stress of the COVID-19 pandemic. However, the advantage of older adults was diminished during the pandemic, pointing to a likely role of older adults use of situation selection strategies (Charles, 2010).

Legislative tools in the Czech watershed management policy

1996 ◽  
Vol 33 (4-5) ◽  
pp. 39-44
Author(s):  
J. Holas ◽  
M. Konvicková
Keyword(s):  
Watershed Management ◽  
Large Scale ◽  
Agricultural Practices ◽  
Farming System ◽  
Management Policy ◽  
Running Water ◽  
The Czech Republic ◽  
Law System ◽  
Water Quality Regulations

Potential environmental impacts as a result of large-scale farming system in the Czech Republic have created a great deal of concern in recent years. This concern has led to several studies to identify the role of new regulations, directives and other legislative issues in the field of water pollution control. The set of legislative tools related to watershed management policy to promote better agricultural practices is shortly reviewed. The paper emphasises the running water law system amendment with respect to European community water quality regulations.

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