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Array-based cross-species hybridization between genera of Rhabditidae (Nematoda)

ABSTRACT: The objective of this work was to determine the effectiveness of cross-hybridization of gDNA from five native soil nematodes to an Affymetrix Caenorhabditis elegans tiling array. Cross-hybridization experiments using C. briggsae, for which genome information is available, allowed hybridisation intensities to be correlated with known sequence differences. Initial analysis of data by conventional array-based Comparative Genomic Hybridization (aCGH) techniques at the chip level lead to misleading results due to an artefact from the combination of scaling, bandwidth smoothing, and differential GC content in exon and intron regions. To circumvent this artefact, individual probes were instead normalized and centered by adjusting for probe-specific thermodynamic binding affinity. However, cross-hybridization of C. briggsae DNA revealed that the resultant probe intensities alone were still uncorrelated to sequence similarity below 90% identity. Below 90% similarity, all probes hybridize uniformly poorly, and above 90% similarity the hybridization differences are not large enough to detect over background, therefore, no 'threshold' ratio of hybridization intensity was successful at identifying probes with similarity to the heterologous genome. In light of the observations described here, we suggest that the criteria for replication and verification of gene expression profiles generated from cross-species microarray hybridizations be more stringent than typically adopted for con-specific hybridizations.

ORGANISM(S): Acrobeloides sp. MAH-2010 Oscheius sp. FVV-2 Caenorhabditis briggsae Chiloplacus sp. MAH-2010 Oscheius tipulae Mesorhabditis sp. MAH-2010 Caenorhabditis elegans

PROVIDER: GSE23667 | GEO | 2010/08/18

SECONDARY ACCESSION(S): PRJNA130825

REPOSITORIES: GEO

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Array-based cross-species hybridization between genera of Rhabditidae (Nematoda)

Project description:The objective of this work was to determine the effectiveness of cross-hybridization of gDNA from five native soil nematodes to an Affymetrix Caenorhabditis elegans tiling array. Cross-hybridization experiments using C. briggsae, for which genome information is available, allowed hybridisation intensities to be correlated with known sequence differences. Initial analysis of data by conventional array-based Comparative Genomic Hybridization (aCGH) techniques at the chip level lead to misleading results due to an artefact from the combination of scaling, bandwidth smoothing, and differential GC content in exon and intron regions. To circumvent this artefact, individual probes were instead normalized and centered by adjusting for probe-specific thermodynamic binding affinity. However, cross-hybridization of C. briggsae DNA revealed that the resultant probe intensities alone were still uncorrelated to sequence similarity below 90% identity. Below 90% similarity, all probes hybridize uniformly poorly, and above 90% similarity the hybridization differences are not large enough to detect over background, therefore, no 'threshold' ratio of hybridization intensity was successful at identifying probes with similarity to the heterologous genome. In light of the observations described here, we suggest that the criteria for replication and verification of gene expression profiles generated from cross-species microarray hybridizations be more stringent than typically adopted for con-specific hybridizations. Genomic DNA from Caenorhabditis elegans N2 (Bristol), C. elegans CB4856 (Hawaiian), C. briggsae AF16, Oscheius tipulae KS585, Oscheius FVV-2 KS555, Mesorhabditis sp. KS587, Acrobeloides sp. KS586, and Chiloplacus sp. KS584 were hybridized onto C. elegans Affymetrix tiling array (two replicate chips were performed for each species).

2010-08-17 | E-GEOD-23667 | biostudies-arrayexpress

Analysis of on-chip hybridization kinetics for optimization of gene expression experiments.

Project description:DNA microarray technology is a powerful tool for getting the overview of gene expression in biological samples. Although the successful application of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover, oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, 15 hours). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray. To validate these hypothesis, on-chip hybridization kinetics and duplexes thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to mouse genes. We demonstrate that differences in hybridization kinetics between the probes can influence the interpretation of expression data. Ways to improve the reliability of microarray-based expression analysis are discussed. Keywords: kinetics, gene expression, microarrays

2007-09-30 | GSE7574 | GEO

C. elegans developmental timecourse

Project description:To identify differences in gene expression patterns between C. elegans and C. briggsae we designed whole-genome species-specific microarrays. We designed two organism-specific microarrays which were then manufactured by Agilent as 44k arrays. Probes were designed to target the coding region, preferentially near the 3’ end, as well as in the presumptive 3’ UTR, up to 150bp downstream of the stop codon. 50-60mer probes were determined using OligoWiz (Wernersson and Nielsen 2005) which selects oligos based upon their cross-hybridization to other coding sequences, Tm, position along the transcript, folding potential, and low-complexity in the sequence. The probes were also restricted against spanning splice junctions to avoid missing transcripts due to errors in gene structure predictions. Each gene was assigned between one and five probes, where the probe with the best overall score was selected as the ‘A-probe’, and the remaining ranked ‘B’ through ‘E’.

2009-08-31 | E-GEOD-15234 | biostudies-arrayexpress

Paralog Specificity in cDNA Microarray Experiments

Project description:cDNA microarrays have become the technology of choice for profiling the expression of thousands of genes simultaneously in a single sample. However, cross-hybridization between targets and probes that do not correspond must be considered carefully during the design, analysis, and interpretation of these experiments. Here, we examined this potentially confounding issue by competitively hybridizing fragments with similar sequence obtained from two paralogous genes to a custom cDNA microarray that contains probes for only one of these two genes. We found that although a large contiguous portion of these two fragments was 84% identical and only one target was represented on the array, there was very little cross-hybridization of the non-represented target to either the paralogous probes or other (non-similar) features. We conclude that cDNA microarrays are indeed very specific and that promiscuous paralog hybridization adds little noise to the data. More generally, the absence of specific probes for any given gene does not increase the chance for cross-hybridization between non-specific probes and targets, even if sequence similarity is relatively high.

2009-07-03 | GSE16939 | GEO

Consensus Preprocessing of GEO and ArrayExpress Normal and Diseased Lung Tissue Microarray Data

Project description:The Affymetrix oligonucleotide microarrays measure gene expression by quantifying intensity of fluorescently labeled gene fragments that bind to sets of 25-mer oligonucleotide probes on the chip with specific sequences tailored to be complementary to the target genes. Each gene is associated with a "probe set" containing several pairs (usually 11) of "perfect match" (perfectly complementary to target sequence) and "mismatch" (different base at position 13 of 25) probes. The raw measurements of each probe set consist of a set of intensities from the probes, which require in silico preprocessing by (1) correcting for background variability, (2) normalizing intensities across samples, and (3) summarizing intensities across the probe set into a single expression value. The output of the summarization step corresponds to the background-adjusted value for the mRNA of interest. We preprocess using GCRMA, which corrects for background variability by accounting for optical noise, probe affinity, and mismatch probe adjustment; normalizes intensities by quantile normalization; and summarizes intensities using a median polish method. To minimize preprocessing batch effects, it is desirable to preprocess all samples in the dataset together. However, preprocessing across multple platforms requires a consolidation of probes with identical sequences, precluding global preprocessing on datasets with multiple platforms using the standard preprocessing pipelines. To address this problem, we have developed and applied a custom preprocessing pipeline to combine the raw .CEL files from multiple platforms that share the same probe sets.

2014-09-18 | GSE60486 | GEO

Control run hybridization

Project description:Laboratory strain poliovirus was hybridized to the array as a control run and a proof of concept. Degree of cross hybridization between polio nucleic acid and non-polio probes was evaluated. Specificity of the probe design was determined. Keywords: control study: target detection and specificity

2008-02-28 | GSE10566 | GEO

Co-hybridization of Adenovirus type 40 and 41

Project description:Co-hybridization of Adenovirus type 40 and 41 to test probe specificity for closely related viral targets. ATCC strains of Adenovirus type 40 and 41 were hybridized to the array as a control run and a proof of concept. Degree of cross hybridization between polio nucleic acid and non-adenovirus 40 and 41 probes was evaluated. Specificity of the probe design was determined between closely related members of the same virus family. Keywords: control study: target detection and specificity

2008-02-28 | GSE10569 | GEO

C. briggsae developmental timecourse

Project description:To identify differences in gene expression patterns between C. elegans and C. briggsae we designed whole-genome species-specific microarrays. We designed two organism-specific microarrays which were then manufactured by Agilent as 44k arrays. Probes were designed to target the coding region, preferentially near the 3â end, as well as in the presumptive 3â UTR, up to 150bp downstream of the stop codon. 50-60mer probes were determined using OligoWiz (Wernersson and Nielsen 2005) which selects oligos based upon their cross-hybridization to other coding sequences, Tm, position along the transcript, folding potential, and low-complexity in the sequence. The probes were also restricted against spanning splice junctions to avoid missing transcripts due to errors in gene structure predictions. Each gene was assigned between one and five probes, where the probe with the best overall score was selected as the âA-probeâ, and the remaining ranked âBâ through âEâ.

2010-05-06 | E-GEOD-15233 | biostudies-arrayexpress

A novel aCGH method for non-model organisms for assessing genomes and de-novo transcriptome sequencing reads (Danio rerio)

Project description:Genomes and transcriptomes of non-model organisms can be analyzed using next-generation sequencing technologies, but de-novo sequencing and annotating a full eukaryotic genome is still challenging. So, -omics experimentation with non-model organisms requires a suite of technologies to obtain reliable results in a cost-effective manner. Here, a novel method for microarray-based genome analysis is presented which is especially suitable for non-model organisms. We show that it is useful for complementing regular aCGH analyses and for evaluating transcriptome next-generation sequencing reads. The principle of the method is straightforward: feature intensities obtained after hybridizing the test genome are compared with the feature intensities of a control hybridization. The control hybridization is performed with negative control probes (no targets in the control sample), and with positive control probes (with targets in the control sample). The method has in principle a resolution of a single probe and it does not depend on the structural information of a reference genome: the genomic ordering of probe targets is irrelevant. In a test, analyzing the genome content of a sequenced bacterial strain: Staphylococcus aureus MRSA252, this approach proved to be successful demonstrated by receiver operating characteristic area under the curve values larger than 0.9995.

2014-05-19 | GSE52121 | GEO

A novel aCGH method for non-model organisms for assessing genomes and de-novo transcriptome sequencing reads (Staphylococcus aureus)

2014-05-19 | GSE51148 | GEO

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