1,480 research outputs found
16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform
peer-reviewedBackground
Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA.
Results
The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar.
Conclusions
Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.This publication has emanated from research supported in part by a research
grant from Science Foundation Ireland (SFI) under Grant Numbers SFI/12/RC/2273 and 11/PI/1137 and by FP7 funded CFMATTERS (Cystic Fibrosis
Microbiome-determined Antibiotic Therapy Trial in Exacerbations: Results Stratified, Grant Agreement no. 603038)
Stack sorting with restricted stacks
The (classical) problem of characterizing and enumerating permutations that
can be sorted using two stacks connected in series is still largely open. In
the present paper we address a related problem, in which we impose restrictions
both on the procedure and on the stacks. More precisely, we consider a greedy
algorithm where we perform the rightmost legal operation (here "rightmost"
refers to the usual representation of stack sorting problems). Moreover, the
first stack is required to be -avoiding, for some permutation ,
meaning that, at each step, the elements maintained in the stack avoid the
pattern when read from top to bottom. Since the set of permutations
which can be sorted by such a device (which we call -machine) is not
always a class, it would be interesting to understand when it happens. We will
prove that the set of -machines whose associated sortable permutations
are not a class is counted by Catalan numbers. Moreover, we will analyze two
specific -machines in full details (namely when and
), providing for each of them a complete characterization and
enumeration of sortable permutations
On modeling and measuring viscoelasticity with dynamic Atomic Force Microscopy
The interaction between a rapidly oscillating atomic force microscope tip and
a soft material surface is described using both elastic and viscous forces with
a moving surface model. We derive the simplest form of this model, motivating
it as a way to capture the impact dynamics of the tip and sample with an
interaction consisting of two components: interfacial or surface force, and
bulk or volumetric force. Analytic solutions to the piece-wise linear model
identify characteristic time constants, providing a physical explanation of the
hysteresis observed in the measured dynamic force quadrature curves. Numerical
simulation is used to fit the model to experimental data and excellent
agreement is found with a variety of different samples. The model parameters
form a dimensionless impact-rheology factor, giving a quantitative physical
number to characterize a viscoelastic surface that does not depend on the tip
shape or cantilever frequency.Comment: 13 pages, 7 figure
Analysis of VEGF-A Regulated Gene Expression in Endothelial Cells to Identify Genes Linked to Angiogenesis
Angiogenesis is important for many physiological processes, diseases, and also regenerative medicine. Therapies that inhibit the vascular endothelial growth factor (VEGF) pathway have been used in the clinic for cancer and macular degeneration. In cancer applications, these treatments suffer from a “tumor escape phenomenon” where alternative pathways are upregulated and angiogenesis continues. The redundancy of angiogenesis regulation indicates the need for additional studies and new drug targets. We aimed to (i) identify novel and missing angiogenesis annotations and (ii) verify their significance to angiogenesis. To achieve these goals, we integrated the human interactome with known angiogenesis-annotated proteins to identify a set of 202 angiogenesis-associated proteins. Across endothelial cell lines, we found that a significant fraction of these proteins had highly perturbed gene expression during angiogenesis. After treatment with VEGF-A, we found increasing expression of HIF-1α, APP, HIV-1 tat interactive protein 2, and MEF2C, while endoglin, liprin β1 and HIF-2α had decreasing expression across three endothelial cell lines. The analysis showed differential regulation of HIF-1α and HIF-2α. The data also provided additional evidence for the role of endothelial cells in Alzheimer's disease
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