664 research outputs found
Specific and non specific hybridization of oligonucleotide probes on microarrays
Gene expression analysis by means of microarrays is based on the sequence
specific binding of mRNA to DNA oligonucleotide probes and its measurement
using fluorescent labels. The binding of RNA fragments involving other
sequences than the intended target is problematic because it adds a "chemical
background" to the signal, which is not related to the expression degree of the
target gene. The paper presents a molecular signature of specific and non
specific hybridization with potential consequences for gene expression
analysis. We analyzed the signal intensities of perfect match (PM) and mismatch
(MM) probes of GeneChip microarrays to specify the effect of specific and non
specific hybridization. We found that these events give rise to different
relations between the PM and MM intensities as function of the middle base of
the PMs, namely a triplet- (C>G=T>A>0) and a duplet-like (C=T>0>G=A) pattern of
the PM-MM log-intensity difference upon binding of specific and non specific
RNA fragments, respectively. The systematic behaviour of the intensity
difference can be rationalized on the level of base pairings of DNA/RNA
oligonucleotide duplexes in the middle of the probe sequence. Non-specific
binding is characterized by the reversal of the central Watson Crick (WC)
pairing for each PM/MM probe pair, whereas specific binding refers to the
combination of a WC and a self complementary (SC) pairing in PM and MM probes,
respectively. The intensity of complementary MM introduces a systematic source
of variation which decreases the precision of expression measures based on the
MM intensities
The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters
Quantifying interactions in DNA microarrays is of central importance for a
better understanding of their functioning. Hybridization thermodynamics for
nucleic acid strands in aqueous solution can be described by the so-called
nearest-neighbor model, which estimates the hybridization free energy of a
given sequence as a sum of dinucleotide terms. Compared with its solution
counterparts, hybridization in DNA microarrays may be hindered due to the
presence of a solid surface and of a high density of DNA strands. We present
here a study aimed at the determination of hybridization free energies in DNA
microarrays. Experiments are performed on custom Agilent slides. The solution
contains a single oligonucleotide. The microarray contains spots with a perfect
matching complementary sequence and other spots with one or two mismatches: in
total 1006 different probe spots, each replicated 15 times per microarray. The
free energy parameters are directly fitted from microarray data. The
experiments demonstrate a clear correlation between hybridization free energies
in the microarray and in solution. The experiments are fully consistent with
the Langmuir model at low intensities, but show a clear deviation at
intermediate (non-saturating) intensities. These results provide new
interesting insights for the quantification of molecular interactions in DNA
microarrays.Comment: 31 pages, 5 figure
Physico-chemical foundations underpinning microarray and next-generation sequencing experiments
Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized
Between-species differences in gene copy number are enriched among functions critical for adaptive evolution in Arabidopsis halleri
Complete dataset expanded from Additional file 3. (XLS 16282 kb
Linear model for fast background subtraction in oligonucleotide microarrays
One important preprocessing step in the analysis of microarray data is
background subtraction. In high-density oligonucleotide arrays this is
recognized as a crucial step for the global performance of the data analysis
from raw intensities to expression values.
We propose here an algorithm for background estimation based on a model in
which the cost function is quadratic in a set of fitting parameters such that
minimization can be performed through linear algebra. The model incorporates
two effects: 1) Correlated intensities between neighboring features in the chip
and 2) sequence-dependent affinities for non-specific hybridization fitted by
an extended nearest-neighbor model.
The algorithm has been tested on 360 GeneChips from publicly available data
of recent expression experiments. The algorithm is fast and accurate. Strong
correlations between the fitted values for different experiments as well as
between the free-energy parameters and their counterparts in aqueous solution
indicate that the model captures a significant part of the underlying physical
chemistry.Comment: 21 pages, 5 figure
G-stack modulated probe intensities on expression arrays - sequence corrections and signal calibration
<p>Abstract</p> <p>Background</p> <p>The brightness of the probe spots on expression microarrays intends to measure the abundance of specific mRNA targets. Probes with runs of at least three guanines (G) in their sequence show abnormal high intensities which reflect rather probe effects than target concentrations. This G-bias requires correction prior to downstream expression analysis.</p> <p>Results</p> <p>Longer runs of three or more consecutive G along the probe sequence and in particular triple degenerated G at its solution end ((<it>GGG</it>)<sub>1</sub>-effect) are associated with exceptionally large probe intensities on GeneChip expression arrays. This intensity bias is related to non-specific hybridization and affects both perfect match and mismatch probes. The (<it>GGG</it>)<sub>1</sub>-effect tends to increase gradually for microarrays of later GeneChip generations. It was found for DNA/RNA as well as for DNA/DNA probe/target-hybridization chemistries. Amplification of sample RNA using T7-primers is associated with strong positive amplitudes of the G-bias whereas alternative amplification protocols using random primers give rise to much smaller and partly even negative amplitudes.</p> <p>We applied positional dependent sensitivity models to analyze the specifics of probe intensities in the context of all possible short sequence motifs of one to four adjacent nucleotides along the 25meric probe sequence. Most of the longer motifs are adequately described using a nearest-neighbor (NN) model. In contrast, runs of degenerated guanines require explicit consideration of next nearest neighbors (GGG terms). Preprocessing methods such as vsn, RMA, dChip, MAS5 and gcRMA only insufficiently remove the G-bias from data.</p> <p>Conclusions</p> <p>Positional and motif dependent sensitivity models accounts for sequence effects of oligonucleotide probe intensities. We propose a positional dependent NN+GGG hybrid model to correct the intensity bias associated with probes containing poly-G motifs. It is implemented as a single-chip based calibration algorithm for GeneChips which can be applied in a pre-correction step prior to standard preprocessing.</p
Real-time DNA microarray analysis
We present a quantification method for affinity-based
DNA microarrays which is based on the
real-time measurements of hybridization kinetics.
This method, i.e. real-time DNA microarrays,
enhances the detection dynamic range of conventional
systems by being impervious to probe
saturation in the capturing spots, washing
artifacts, microarray spot-to-spot variations, and
other signal amplitude-affecting non-idealities. We
demonstrate in both theory and practice that the
time-constant of target capturing in microarrays,
similar to all affinity-based biosensors, is inversely
proportional to the concentration of the target
analyte, which we subsequently use as the fundamental
parameter to estimate the concentration
of the analytes. Furthermore, to empirically
validate the capabilities of this method in practical
applications, we present a FRET-based assay which
enables the real-time detection in gene expression
DNA microarrays
Human Leukocyte Antigen Typing Using a Knowledge Base Coupled with a High-Throughput Oligonucleotide Probe Array Analysis
Human leukocyte antigens (HLA) are important biomarkers because multiple diseases, drug toxicity, and vaccine responses reveal strong HLA associations. Current clinical HLA typing is an elimination process requiring serial testing. We present an alternative in situ synthesized DNA-based microarray method that contains hundreds of thousands of probes representing a complete overlapping set covering 1,610 clinically relevant HLA class I alleles accompanied by computational tools for assigning HLA type to 4-digit resolution. Our proof-of-concept experiment included 21 blood samples, 18 cell lines, and multiple controls. The method is accurate, robust, and amenable to automation. Typing errors were restricted to homozygous samples or those with very closely related alleles from the same locus, but readily resolved by targeted DNA sequencing validation of flagged samples. High-throughput HLA typing technologies that are effective, yet inexpensive, can be used to analyze the world’s populations, benefiting both global public health and personalized health care
Motif effects in Affymetrix GeneChips seriously affect probe intensities
An Affymetrix GeneChip consists of an array of hundreds of thousands of probes (each a sequence of 25 bases) with the probe values being used to infer the extent to which genes are expressed in the biological material under investigation. In this article, we demonstrate that these probe values are also strongly influenced by their precise base sequence. We use data from >28 000 CEL files relating to 10 different Affymetrix GeneChip platforms and involving nearly 1000 experiments. Our results confirm known effects (those due to the T7-primer and the formation of G-quadruplexes) but reveal other effects. We show that there can be huge variations from one experiment to another, and that there may also be sizeable disparities between batches within an experiment and between CEL files within a batch. © 2012 The Author(s)
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