Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements

Abstract Background Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products.

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Ligation bias is a major contributor to nonstoichiometric abundances of secondary siRNAs and impacts analyses of microRNAs

10.1101/2020.09.14.296616 ◽

2020 ◽

Author(s):

Patricia Baldrich ◽

Saleh Tamim ◽

Sandra Mathioni ◽

Blake Meyers

Keyword(s):

Small Rna ◽

Small Rnas ◽

High Throughput Sequencing ◽

Library Construction ◽

Construction Methods ◽

Wide Range ◽

Secondary Sirnas ◽

Two Stages ◽

Material Library ◽

Small Rna Library

ABSTRACTPlant small RNAs are a diverse and complex set of molecules, ranging in length from 21 to 24 nt, involved in a wide range of essential biological processes. High-throughput sequencing is used for the discovery and quantification of small RNAs. However, several biases can occur during the preparation of small RNA libraries, especially using low input RNA. We used two stages of maize anthers to evaluate the performance of seven commercially-available methods for small RNA library construction, using different RNA input amounts. We show that when working with plant material, library construction methods have differing capabilities to capture small RNAs, and that different library construction methods provide better results when applied to the detection of microRNAs, phasiRNAs, or tRNA-derived fragment. We also observed that ligation bias occurs at both ends of miRNAs and phasiRNAs, suggesting that the biased compositions observed in small RNA populations, including nonstoichiometric levels of phasiRNAs within a locus, may reflect a combination of biological and technical influences.

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Colorimetric research of self-luminous objects in software and hardware environments by the method of multidimensional scales implementation

Izmeritel`naya Tekhnika ◽

10.32446/0368-1025it.2020-8-43-50 ◽

2020 ◽

pp. 43-50

Author(s):

Yauheniya N. Saukova

Keyword(s):

Metrological Traceability ◽

Point Of View ◽

Intermediate Precision ◽

Technical Vision ◽

Wide Range ◽

Chromaticity Coordinates ◽

Unity Of Measurements ◽

Software And Hardware ◽

Luminous Objects ◽

Reference Samples

It is shown that the issues of metrological traceability for extended self-luminous objects with a wide range of brightness have not yet been resolved, since the rank scales of embedded systems are used for processing digital images. For such scales, there is no “fixed” unit, which does not allow you to get reliable results and ensure the unity of measurements. An experiment is described to evaluate the accuracy of determining the intensity (coordinates) of the color of self-luminous objects. In terms of repeatability and intermediate precision compared to the reference measurement method, the color and chromaticity coordinates of self-luminous objects (reference samples) were determined by their multiple digital registration using technical vision systems. The possibilities of the developed methodology for colorimetric studies in hardware and software environments from the point of view of constructing a multidimensional conditional scale are determined.

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Extraction of DNA from a Wide Range of Objects by Treatment with Ammonium Salts

Biotekhnologiya ◽

10.21519/0234-2758-2020-36-6-98-106 ◽

2020 ◽

Vol 36 (6) ◽

pp. 98-106

Author(s):

E.I. Levitin ◽

B.V. Sviridov ◽

O.V. Piksasova ◽

T.E. Shustikova

Keyword(s):

Dna Extraction ◽

Dna Isolation ◽

Tissue Samples ◽

New Approach ◽

Cell Wall Disruption ◽

Wide Range ◽

Low Salt ◽

Mammalian Blood ◽

Plasmid Extraction ◽

Two Hybrid

Currently, simple, rapid, and efficient techniques for DNA isolation from a wide range of organisms are in demand in biotechnology and bioinformatics. A key (and often limiting) step is the cell wall disruption and subsequent DNA extraction from the disintegrated cells. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: treatment of cells or tissue samples with ammonium acetate followed by cell lysis in low-salt buffer with the addition of SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for high-molecular native DNA isolation from bacteria, ascomycetes, yeast, and mammalian blood; it is also useful for express analysis of environmental microbial isolates and for plasmid extraction for two-hybrid library screening. express method for DNA isolation; ammonium salt treatment (в русских ключевых такой порядок), osmotic breakage of cells This study was financially supported by the NRC "Kurchatov Institute"-GOSNIIGENETIKA Kurchatov Genomic Center.

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Identification of an N-acetylneuraminic acid-presenting bacteria isolated from a human microbiome

Scientific Reports ◽

10.1038/s41598-021-83875-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhen Han ◽

Peter S. Thuy-Boun ◽

Wayne Pfeiffer ◽

Vincent F. Vartabedian ◽

Ali Torkamani ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Human Microbiome ◽

Host Immunity ◽

Rrna Gene ◽

Metabolic Labeling ◽

Selective Labeling ◽

Fecal Microbiome ◽

Acetylneuraminic Acid ◽

Intestinal Mucus ◽

Microbial Systems

AbstractN-Acetylneuraminic acid is the most abundant sialic acid (SA) in humans and is expressed as the terminal sugar on intestinal mucus glycans. Several pathogenic bacteria harvest and display host SA on their own surfaces to evade Siglec-mediated host immunity. While previous studies have identified bacterial enzymes associated with SA catabolism, no reported methods permit the selective labeling, tracking, and quantitation of SA-presenting microbes within complex multi-microbial systems. We combined metabolic labeling, click chemistry, 16S rRNA gene, and whole-genome sequencing to track and identify SA-presenting microbes from a cultured human fecal microbiome. We isolated a new strain of Escherichia coli that incorporates SA onto its own surface and encodes for the nanT, neuA, and neuS genes necessary for harvesting and presenting SA. Our method is applicable to the identification of SA-presenting bacteria from human, animal, and environmental microbiomes, as well as providing an entry point for the investigation of surface-expressed SA-associated structures.

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Gene Expression Space Shapes the Bioprocess Trade-Offs among Titer, Yield and Productivity

Applied Sciences ◽

10.3390/app11135859 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5859

Author(s):

Fernando N. Santos-Navarro ◽

Yadira Boada ◽

Alejandro Vignoni ◽

Jesús Picó

Keyword(s):

Gene Expression ◽

Gene Transcription ◽

Performance Metrics ◽

Cell Mass ◽

Gene Copy ◽

Substrate Uptake ◽

Promoter Strength ◽

Expression Levels ◽

Trade Offs ◽

Wide Range

Optimal gene expression is central for the development of both bacterial expression systems for heterologous protein production, and microbial cell factories for industrial metabolite production. Our goal is to fulfill industry-level overproduction demands optimally, as measured by the following key performance metrics: titer, productivity rate, and yield (TRY). Here we use a multiscale model incorporating the dynamics of (i) the cell population in the bioreactor, (ii) the substrate uptake and (iii) the interaction between the cell host and expression of the protein of interest. Our model predicts cell growth rate and cell mass distribution between enzymes of interest and host enzymes as a function of substrate uptake and the following main lab-accessible gene expression-related characteristics: promoter strength, gene copy number and ribosome binding site strength. We evaluated the differential roles of gene transcription and translation in shaping TRY trade-offs for a wide range of expression levels and the sensitivity of the TRY space to variations in substrate availability. Our results show that, at low expression levels, gene transcription mainly defined TRY, and gene translation had a limited effect; whereas, at high expression levels, TRY depended on the product of both, in agreement with experiments in the literature.

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Design and Evaluation of Large-Scale IoT-Enabled Healthcare Architecture

Applied Sciences ◽

10.3390/app11083623 ◽

2021 ◽

Vol 11 (8) ◽

pp. 3623

Author(s):

Omar Said ◽

Amr Tolba

Keyword(s):

Packet Loss ◽

Large Scale ◽

Performance Metrics ◽

Medical System ◽

Healthcare Data ◽

High Altitude Platforms ◽

Wide Range ◽

Simulation Results ◽

Healthcare Architecture ◽

The Internet Of Things

Employment of the Internet of Things (IoT) technology in the healthcare field can contribute to recruiting heterogeneous medical devices and creating smart cooperation between them. This cooperation leads to an increase in the efficiency of the entire medical system, thus accelerating the diagnosis and curing of patients, in general, and rescuing critical cases in particular. In this paper, a large-scale IoT-enabled healthcare architecture is proposed. To achieve a wide range of communication between healthcare devices, not only are Internet coverage tools utilized but also satellites and high-altitude platforms (HAPs). In addition, the clustering idea is applied in the proposed architecture to facilitate its management. Moreover, healthcare data are prioritized into several levels of importance. Finally, NS3 is used to measure the performance of the proposed IoT-enabled healthcare architecture. The performance metrics are delay, energy consumption, packet loss, coverage tool usage, throughput, percentage of served users, and percentage of each exchanged data type. The simulation results demonstrate that the proposed IoT-enabled healthcare architecture outperforms the traditional healthcare architecture.

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Nonlinear dynamic behaviour of guy cables in turbulent winds

Canadian Journal of Civil Engineering ◽

10.1139/l00-089 ◽

2001 ◽

Vol 28 (1) ◽

pp. 98-110 ◽

Cited By ~ 4

Author(s):

Bruce F Sparling ◽

Alan G Davenport

Keyword(s):

Finite Element ◽

Dynamic Behaviour ◽

Aerodynamic Drag ◽

Numerical Study ◽

Horizontal Displacement ◽

Wind Loading ◽

Second Phase ◽

Nonlinear Coupling ◽

Wide Range ◽

Cable Vibrations

Large amplitude cable vibrations are difficult to predict using linear theory due to the presence of sag in the suspended profile. A numerical study was therefore undertaken to investigate the dynamic behaviour of inclined cables excited by imposed displacements. To model the nonlinear nature of cable response, a time domain finite element approach was adopted using nonlinear catenary cable elements. Two types of horizontal displacement patterns were enforced at the upper end of the guy. In the first phase of the study, harmonic displacement histories with a wide range of forcing frequencies were considered. In the second phase, random enforced displacements were used to simulate the motion of a guyed mast in gusty winds. The influence of aerodynamic drag and damping forces was investigated by performing analyses under still air, steady wind, and turbulent wind conditions. It was found that nonlinear coupling of related harmonic response components was significant at certain critical frequencies, particular when the excitation was harmonic and acted in the plane of the guy. Positive aerodynamic damping was shown to effectively suppress resonant and nonlinear coupling response.Key words: cables, structural dynamics, wind loading, finite element method, nonlinear analysis, guyed towers.

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Abstract TP132: Selection and Activation of Sites in a Large Multi-Center Randomized Clinical Trial

Stroke ◽

10.1161/str.48.suppl_1.tp132 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Bart M Demaerschalk ◽

Robert D Brown ◽

Virginia J Howard ◽

MeeLee Tom ◽

Mary E Longbottom ◽

...

Keyword(s):

Site Selection ◽

Performance Metrics ◽

Interventional Neuroradiology ◽

Interquartile Range ◽

Carotid Revascularization ◽

Training Requirements ◽

Medical Centers ◽

Wide Range ◽

The Times ◽

And Training

Introduction: Careful selection and timely activation of clinical sites in multicenter clinical trials is critical for successful enrollment, subject safety, and generalizability of results. Methods: In the Carotid Revascularization and Medical Management for Asymptomatic Carotid Stenosis Trial (CREST-2), a multidisciplinary Site Selection Committee evaluated applicants referred via participation in CREST, CREST principal investigators (PIs) and other investigators, StrokeNet and industry partners. Data for consideration included performance metrics in CREST and other carotid trials and a site selection questionnaire containing information on the investigators as well as quantitative data on carotid procedures performed. Any FDA warning letters were reviewed. Results: The Committee met bi-weekly for 36 months (n=64 meetings). Applications from 176 sites between March 2014 and July 2016 were evaluated: 153 were approved, 7 are under Committee review, 5 were approved but withdrew, 5 were placed on a waiting list, and 6 were rejected. One-hundred-four sites have completed the regulatory and training requirements to randomize: 51 (49%) academic medical centers, 31 (30%) private hospital-based centers, 16 (15%) private office-based practices, and 6 (6%) Veterans Administration medical centers. The mean times from application-to- approval was 5.2 weeks (interquartile range, 1.9, 6.2), and from approval-to-randomization status was 46.7 weeks (interquartile range, 35.4, 51.7). Specialties of the 104 site PIs are vascular surgery for 35 (33.7%), cardiology for 30 (28.8%), neurology for 25 (24%), neurosurgery for 8 (7.7%), interventional radiology for 4 (3.8%), and interventional neuroradiology for 2 (1.9%). Conclusions: Careful site selection is time-consuming for prospective sites and for trial leadership. Times from application-to-site-approval were modest (mean = 5.2 weeks), in contrast to the times for completing regulatory and training requirements (mean = 46.7 weeks). However, subject enrollment by teams from a wide range of medical centers led by a multi-disciplinary cohort of PIs will promote the generalizability of trial results.

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A protocol for an intercomparison of biodiversity and ecosystem services models using harmonized land-use and climate scenarios

Geoscientific Model Development ◽

10.5194/gmd-11-4537-2018 ◽

2018 ◽

Vol 11 (11) ◽

pp. 4537-4562 ◽

Cited By ~ 25

Author(s):

HyeJin Kim ◽

Isabel M. D. Rosa ◽

Rob Alkemade ◽

Paul Leadley ◽

George Hurtt ◽

...

Keyword(s):

Climate Change ◽

Land Use ◽

Ecosystem Services ◽

Biological Diversity ◽

Multiple Scales ◽

Convention On Biological Diversity ◽

Second Phase ◽

Intergovernmental Panel ◽

Wide Range ◽

Global Impacts

Abstract. To support the assessments of the Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES), the IPBES Expert Group on Scenarios and Models is carrying out an intercomparison of biodiversity and ecosystem services models using harmonized scenarios (BES-SIM). The goals of BES-SIM are (1) to project the global impacts of land-use and climate change on biodiversity and ecosystem services (i.e., nature's contributions to people) over the coming decades, compared to the 20th century, using a set of common metrics at multiple scales, and (2) to identify model uncertainties and research gaps through the comparisons of projected biodiversity and ecosystem services across models. BES-SIM uses three scenarios combining specific Shared Socio-economic Pathways (SSPs) and Representative Concentration Pathways (RCPs) – SSP1xRCP2.6, SSP3xRCP6.0, SSP5xRCP8.6 – to explore a wide range of land-use change and climate change futures. This paper describes the rationale for scenario selection, the process of harmonizing input data for land use, based on the second phase of the Land Use Harmonization Project (LUH2), and climate, the biodiversity and ecosystem services models used, the core simulations carried out, the harmonization of the model output metrics, and the treatment of uncertainty. The results of this collaborative modeling project will support the ongoing global assessment of IPBES, strengthen ties between IPBES and the Intergovernmental Panel on Climate Change (IPCC) scenarios and modeling processes, advise the Convention on Biological Diversity (CBD) on its development of a post-2020 strategic plans and conservation goals, and inform the development of a new generation of nature-centred scenarios.

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Resource allocation and task admission control in cloud systems

10.32920/ryerson.14645862.v1 ◽

2021 ◽

Author(s):

Haleh Khojasteh

Keyword(s):

Resource Allocation ◽

Performance Metrics ◽

Cloud Model ◽

Blocking Probability ◽

Arrival Rate ◽

Cloud System ◽

Wide Range ◽

Priority Scheme ◽

The Impact ◽

And Task

The focus of this thesis is solving the problem of resource allocation in cloud datacenter using an Infrastructure-as-a-Service (IaaS) cloud model. We have investigated the behavior of IaaS cloud datacenters through detailed analytical and simulation models that model linear, transitional and saturated operation regimes. We have obtained accurate performance metrics such as task blocking probability, total delay, utilization and energy consumption. Our results show that the offered load does not offer complete characterization of datacenter operation; therefore, in our evaluations, we have considered the impact of task arrival rate and task service time separately. To keep the cloud system in the linear operation regime, we have proposed several dynamic algorithms to control the admission of incoming tasks. In our first solution, task admission is based on task blocking probability and predefined thresholds for task arrival rate. The algorithms in our second solution are based on full rate task acceptance threshold and filtering coefficient. Our results confirm that the proposed task admission mechanisms are capable of maintaining the stability of cloud system under a wide range of input parameter values. Finally, we have developed resource allocation solutions for mobile clouds in which offloading requests from a mobile device can lead to forking of new tasks in on-demand manner. To address this problem, we have proposed two flexible resource allocation mechanisms with different prioritization: one in which forked tasks are given full priority over newly arrived ones, and another in which a threshold is established to control the priority. Our results demonstrate that threshold-based priority scheme presents better system performance than the full priority scheme. Our proposed solution for clouds with mobile users can be also applied in other clouds which their users’ applications fork new tasks.

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