Limited added value of fungal ITS amplicon sequencing in the study of bovine abortion

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

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mixMC: a multivariate statistical framework to gain insight into Microbial Communities

10.1101/044206 ◽

2016 ◽

Cited By ~ 3

Author(s):

Kim-Anh Le Cao ◽

Mary-Ellen Costello ◽

Vanessa Anne Lakis ◽

Francois Bartolo ◽

Xin-Yi Chua ◽

...

Keyword(s):

Microbial Communities ◽

Biomarker Discovery ◽

Amplicon Sequencing ◽

Added Value ◽

Statistical Tools ◽

Multivariate Statistical ◽

Shotgun Metagenomics ◽

16S Rrna Amplicon Sequencing ◽

Microbial Biomarkers ◽

Culture Independent

Culture independent techniques, such as shotgun metagenomics and 16S rRNA amplicon sequencing have dramatically changed the way we can examine microbial communities. Recently, changes in microbial community structure and dynamics have been associated with a growing list of human diseases. The identification and comparison of bacteria driving those changes requires the development of sound statistical tools, especially if microbial biomarkers are to be used in a clinical setting. We present mixMC, a novel multivariate data analysis framework for metagenomic biomarker discovery. mixMC accounts for the compositional nature of 16S data and enables detection of subtle differences when high inter-subject variability is present due to microbial sampling performed repeatedly on the same subjects but in multiple habitats. Through data dimension reduction the multivariate methods provide insightful graphical visualisations to characterise each type of environment in a detailed manner. We applied mixMC to 16S microbiome studies focusing on multiple body sites in healthy individuals, compared our results with existing statistical tools and illustrated added value of using multivariate methodologies to fully characterise and compare microbial communities.

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Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data

10.1101/213470 ◽

2017 ◽

Cited By ~ 2

Author(s):

Jonathan M Palmer ◽

Michelle A Jusino ◽

Mark T Banik ◽

Daniel L Lindner

Keyword(s):

Data Analysis ◽

Amplicon Sequencing ◽

Variable Length ◽

Its Sequences ◽

Synthetic Control ◽

Mock Community ◽

Software Pipeline ◽

Sequencing Platforms ◽

Fungal Its ◽

Mock Communities

High throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal ITS amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community (BioMock), consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: 1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, 2) pre-clustering steps for variable length amplicons are critically important, 3) a major source of bias is attributed to initial PCR reactions and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological synthetic mock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality.

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The Added Value of Graphical Input and Display for Electron Lens Design

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179701 ◽

1990 ◽

Vol 48 (1) ◽

pp. 190-191

Author(s):

B. Lencova ◽

G. Wisselink

Keyword(s):

Flux Density ◽

Numerical Data ◽

Added Value ◽

Lens Design ◽

Step Size ◽

Magnetic Lens ◽

Flux Lines ◽

Fine Mesh ◽

Electron Lens ◽

User Friendly

Recent progress in computer technology enables the calculation of lens fields and focal properties on commonly available computers such as IBM ATs. If we add to this the use of graphics, we greatly increase the applicability of design programs for electron lenses. Most programs for field computation are based on the finite element method (FEM). They are written in Fortran 77, so that they are easily transferred from PCs to larger machines.The design process has recently been made significantly more user friendly by adding input programs written in Turbo Pascal, which allows a flexible implementation of computer graphics. The input programs have not only menu driven input and modification of numerical data, but also graphics editing of the data. The input programs create files which are subsequently read by the Fortran programs. From the main menu of our magnetic lens design program, further options are chosen by using function keys or numbers. Some options (lens initialization and setting, fine mesh, current densities, etc.) open other menus where computation parameters can be set or numerical data can be entered with the help of a simple line editor. The "draw lens" option enables graphical editing of the mesh - see fig. I. The geometry of the electron lens is specified in terms of coordinates and indices of a coarse quadrilateral mesh. In this mesh, the fine mesh with smoothly changing step size is calculated by an automeshing procedure. The options shown in fig. 1 allow modification of the number of coarse mesh lines, change of coordinates of mesh points or lines, and specification of lens parts. Interactive and graphical modification of the fine mesh can be called from the fine mesh menu. Finally, the lens computation can be called. Our FEM program allows up to 8000 mesh points on an AT computer. Another menu allows the display of computed results stored in output files and graphical display of axial flux density, flux density in magnetic parts, and the flux lines in magnetic lenses - see fig. 2. A series of several lens excitations with user specified or default magnetization curves can be calculated and displayed in one session.

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Preliminary Findings on the Use of Session Questionnaires to Improve Clinician-Client Alliance With Special Consideration to Clinical Education

Perspectives on Administration and Supervision ◽

10.1044/aas25.1.50 ◽

2015 ◽

Vol 25 (1) ◽

pp. 50-60

Author(s):

Anu Subramanian

Keyword(s):

Graduate Students ◽

Clinical Education ◽

Clinical Decision Making ◽

Rating Scale ◽

Treatment Effectiveness ◽

Clinical Decision ◽

Added Value ◽

Therapeutic Outcomes ◽

Specific Feedback ◽

Stakeholder Perspective

ASHA's focus on evidence-based practice (EBP) includes the family/stakeholder perspective as an important tenet in clinical decision making. The common factors model for treatment effectiveness postulates that clinician-client alliance positively impacts therapeutic outcomes and may be the most important factor for success. One strategy to improve alliance between a client and clinician is the use of outcome questionnaires. In the current study, eight parents of toddlers who attended therapy sessions at a university clinic responded to a session outcome questionnaire that included both rating scale and descriptive questions. Six graduate students completed a survey that included a question about the utility of the questionnaire. Results indicated that the descriptive questions added value and information compared to using only the rating scale. The students were varied in their responses regarding the effectiveness of the questionnaire to increase their comfort with parents. Information gathered from the questionnaire allowed for specific feedback to graduate students to change behaviors and created opportunities for general discussions regarding effective therapy techniques. In addition, the responses generated conversations between the client and clinician focused on clients' concerns. Involving the stakeholder in identifying both effective and ineffective aspects of therapy has advantages for clinical practice and education.

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577 Recognising concomitant heart failure in elderly patients with stable COPD: added value of NTproBNP?

European Journal of Heart Failure Supplements ◽

10.1016/s1567-4215(05)80353-4 ◽

2005 ◽

Vol 4 (1) ◽

pp. 129-129

Keyword(s):

Heart Failure ◽

Elderly Patients ◽

Added Value

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The Added Value of RxP Training

PsycEXTRA Dataset ◽

10.1037/e565742006-004 ◽

2003 ◽

Author(s):

John L. Caccavale

Keyword(s):

Added Value

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Crisis Reactions and Their Consequences for the Real Sector of the Russian Economy

Voprosy Ekonomiki ◽

10.32609/0042-8736-2011-1-39-50 ◽

2011 ◽

pp. 39-50

Author(s):

V. Lushin

Keyword(s):

Financial Crisis ◽

Acute Phase ◽

Dutch Disease ◽

Added Value ◽

Real Sector ◽

The Real ◽

Balanced Development ◽

Russian Economy ◽

The Government ◽

State Modernization

The author analyzes factors that led to a deeper fall in output and profitability in the real sector of the Russian economy in comparison with other segments during the acute phase of the financial crisis. It is argued that some contradictions in the government anti-recession policy, activities of the financial sector and natural monopolies lead to pumping out added value created in manufacturing and agriculture, increase symptoms of the «Dutch disease», etc. It is shown that it may threaten the balanced development of the Russian economy, and a set of measures is suggested to minimize these tendencies and create a basis for the state modernization policy.

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