ABSTRACT
New tools for reproducible exploratory data analysis of large datasets are important to address the rising size and complexity of genomic data. We developed the valr R package to enable flexible and efficient genomic interval analysis. valr leverages new tools available in the "tidyverse", including dplyr. Benchmarks of valr show it performs similar to BEDtools and can be used for interactive analyses and incorporated into existing analysis pipelines.
ABSTRACT
Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG) and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemimethylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the K(D) for 1xMBD-GFP ranged from 106 to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The K(D) values for nonsymmetrical methylation motifs were consistently greater (>2 muM), but the binding selectivity between symmetric and hemimethylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density.
Subject(s)
DNA Methylation , DNA-Binding Proteins/metabolism , DNA/analysis , Oligonucleotide Array Sequence Analysis/methods , Surface Plasmon Resonance/methods , Base Sequence , CpG Islands , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sensitivity and SpecificityABSTRACT
Combining a modified two-step chemical etch method with equations to predict etch parameters and photon-plasmon phase-matching resulted in single-mode tapered optical fibers (TOFs) to optimize electromagnetic field enhancement. The phase-matching equation was used to identify the angle of incidence near the TOF cutoff radius at which surface plasmon resonance (SPR) is maximized. The axisymmetric Young-Laplace equation was used to predict the angle of incidence from the fabrication of a TOF via chemical etching. An optimal cone angle of 20.0 degrees , angles of incidence averaging (81.6 +/- 1.9) degrees , and tip diameters of (80.0 +/- 14.1) nm were achieved through a two-step etching process. These TOF characteristics maximize SPR excitation and field enhancement. The refractive index for optimized SPR excitation in the fabricated TOFs at a wavelength of 650 nm was found to be 1.343.
