Large scale analysis of positional effects of single-base mismatches on microarray gene expression data

<p>Abstract</p> <p>Background</p> <p>Affymetrix GeneChips utilize 25-mer oligonucleotides probes linked to a silica surface to detect targets in solution. Mismatches due to single nucleotide polymorphisms (SNPs) can affect the hybridization between probes and targets. Previous research has indicated that binding between probes and targets strongly depends on the positions of these mismatches. However, there has been substantial variability in the effect of mismatch type across studies.</p> <p>Methods</p> <p>By taking advantage of naturally occurring mismatches between rhesus macaque transcripts and human probes from the Affymetrix U133 Plus 2 GeneChip, we collected the largest 25-mer probes dataset with single-base mismatches at each of the 25 positions on the probe ever used in this type of analysis.</p> <p>Results</p> <p>A mismatch at the center of a probe led to a greater loss in signal intensity than a mismatch at the ends of the probe, regardless of the mismatch type. There was a slight asymmetry between the ends of a probe: effects of mismatches at the 3' end of a probe were greater than those at the 5' end. A cross study comparison of the effect of mismatch types revealed that results were not in good agreement among different reports. However, if the mismatch types were consolidated to purine or pyrimidine mismatches, cross study conclusions could be generated.</p> <p>Conclusion</p> <p>The comprehensive assessment of the effects of single-base mismatches on microarrays provided in this report can be useful for improving future versions of microarray platform design and the corresponding data analysis algorithms.</p

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

Free energy of DNA duplex formation on short oligonucleotide microarrays

DNA/DNA duplex formation is the basic mechanism that is used in genome tiling arrays and SNP arrays manufactured by Affymetrix. However, detailed knowledge of the physical process is still lacking. In this study, we show a free energy analysis of DNA/DNA duplex formation these arrays based on the positional-dependent nearest-neighbor (PDNN) model, which was developed previously for describing DNA/RNA duplex formation on expression microarrays. Our results showed that the two ends of a probe contribute less to the stability of the duplexes and that there is a microarray surface effect on binding affinities. We also showed that free energy cost of a single mismatch depends on the bases adjacent to the mismatch site and obtained a comprehensive table of the cost of a single mismatch under all possible combination of adjacent bases. The mismatch costs were found to be correlated with those determined in aqueous solution. We further demonstrate that the DNA copy number estimated from the SNP array correlates negatively with the target length; this is presumably caused by inefficient PCR amplification for long fragments. These results provide important insights into the molecular mechanisms of microarray technology and have implications for microarray design and the interpretation of observed data

Position dependent mismatch discrimination on DNA microarrays – experiments and model

<p>Abstract</p> <p>Background</p> <p>The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.</p> <p>Results</p> <p>We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends) as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results.</p> <p>Conclusion</p> <p>Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN) can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.</p

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>)₁-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>)₁-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

Sequence dependence of cross-hybridization on short oligo microarrays

One of the critical problems in the short oligo microarray technology is how to deal with cross-hybridization that produces spurious data. Little is known about the details of cross-hybridization effect at molecular level. Here, we report a free energy analysis of cross-hybridization on short oligo microarrays using data from a spike-in study. Our analysis revealed that cross-hybridization on the arrays is mostly caused by oligo fragments with a run of 10–16 nt complementary to the probes. Mismatches were estimated to be energetically much more costly in cross-hybridization than that in gene-specific hybridization, implying that the sources of cross-hybridization must be very different between a PM–MM probe pair. Consequently, it is unreliable to use MM probe signal to track cross-hybridizing signal on a corresponding PM probe. Our results also showed that the oligo fragments tend to bind to the 5′ ends of the probes, and are rarely seen at the 3′ ends. These results are useful for microarray design and data analysis

A Functional Gene Array for Detection of Bacterial Virulence Elements

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples

Sequence specific probe signals on SNP microarrays

Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. This thesis contemplates the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates

Combining induced protease fragment assembly and microarray analysis to monitor signaling in living cells.

Die Fähigkeit Signalkaskaden zu vermessen ist für das Verständnis komplexer biologischer Prozesse essentiell. Bis jetzt versorgt uns die DNA Microarray Technologie mit umfassenden Daten, deren Auflösung jedoch auf der Ebene der Genexpression endet. Diese Informationen reichen nicht aus um die vorgeschalteten regulatorischen Mechanismen der Genexpression zu verstehen. Die meisten proteomischen Technologien hängen von in vitro synthetisierten Peptiden ab oder benötigen weitere biochemische Manipulationen. Für die Charakterisierung und Beobachtung einzelner Bestandteile von Signalkaskaden in lebenden Zellen sind Hochdurchsatz-Verfahren notwendig. In der vorliegenden Arbeit wird ein experimentelles Verfahren namens EXTassay beschrieben, dass eine quantitative und parallele Messung multipler Signal-Ereignisse ermöglicht, die der mRNA Expression vorgelagert sind. EXTassays vereinen verschiedene zelluläre Assays, die an die Reporter Gen Expression gekoppelt sind. Um Multiplexing zu erreichen wurde eine komplexe und optimierte Bibliotek kurzer expressed oligonucleotide tags (EXTs) generiert. Jedes einzelne EXT ersetzt hierbei ein klassisches Reportergen und dient als eindeutiger Identifikator für einen definierten zellulären Assay. Es können verschiedene EXTs, die entweder in einer Zelle oder in einer Zellpopulation exprimiert sein können, über Microarray Hybridisierung analysiert werden. In dieser Arbeit wurden Protokolle für das verlässliche Auslesen von Microarrays für EXTs optimiert. Weiterhin wurden EXT-basierte Assays verwendet, um die durch Neuregulin-1 induzierte Dimerisierung und Aktivierung von Rezeptortyrosinkinasen der ErbB Familie zu untersuchen. Für die quantitative Messung von Rezeptordimeriserung und phosphorylationsabhängige Kopplung an Interaktionspartner wurden Protein-Komplementations-Assays der TEV Protease, split-TEV Assays, verwendet. Hierzu wurde jeder Assay an eindeutige EXT-Reporter gekoppelt. Zusätzlich wurde die Aktivierung von 30 verschiedenen EXT-gekoppelten cis-regulatorischen Elementen erfaßt, um so einen Einblick in die nachfolgende Aspekte der Signalverarbeitung zu erhalten. Alle Assays wurden mit eindeutigen EXTs durchgeführt und mittels Microarray analysiert. Die simultane Analyse dreier verschiedener und regulierter Rezeptor Komplexe (ErbB2/2, 2/3, 2/4) zeigte, dass EXT-basierte Assays geeignet sind rezeptor-spezifische Signalereignisse zu unterscheiden. EXTassays sind daher geeignet quantitative Profile aktivierter Signalkaskaden in Zellen erstellen zu können.The ability to monitor multiple signaling events simultaneously in living cells is essential to better understand complex biological processes. So far, DNA-microarray technologies provide global scale data mainly restricted to the level of gene expression. This information is not sufficient to understand the upstream regulatory mechanisms that lead to gene expression changes. Most proteomic technologies also provide large scale measurement but usually depend on in vitro synthesized peptides or require biochemical manipulations. High throughput technologies are required for functional characterization and monitoring of signaling components in living cells. Here, an experimental approach is presented termed EXTassay that enables quantitative and parallel measurements of various signaling events upstream of mRNA expression. EXTassay incorporates various cellular assays that are coupled to reporter gene expression. To achieve multiplexing, we have generated a complex and optimized library of short expressed oligonucleotide tags (EXTs). Each unique EXT can replace a classical reporter gene and serves as a unique identifier for tracking and quantification of a defined cellular assay. Multiple EXT-reporters expressed in the same cell or cell population can be isolated and analyzed by custom microarray hybridization. We have established protocols and optimized the microarray readout for reliable EXT quantification. We applied the EXTassay to analyze the neuregulin 1 induced ErbB receptor tyrosine kinase signaling in PC-12 cells. We used transcriptionally coupled split TEV protein complementation assays to monitor ErbB receptor dimerization and phosphorylation dependent interaction with downstream signaling proteins. In addition, we employed 30 different cis-regulatory elements to assess the downstream signaling. All assays were coupled to unique EXTs and analyzed by microarrays. By analyzing three different receptor complexes (ErbB 2/2, 2/3 and 2/4), we were able to measure receptor specific differential signaling effects and demonstrate that EXTassays can be applied for the quantitative profiling of activated signaling pathways