Dataset Information

Model-based extension of high-throughput to high-content data.

ABSTRACT:

Background

High-quality quantitative data is a major limitation in systems biology. The experimental data used in systems biology can be assigned to one of the following categories: assays yielding average data of a cell population, high-content single cell measurements and high-throughput techniques generating single cell data for large cell populations. For modeling purposes, a combination of data from different categories is highly desirable in order to increase the number of observable species and processes and thereby maximize the identifiability of parameters.

Results

In this article we present a method that combines the power of high-content single cell measurements with the efficiency of high-throughput techniques. A calibration on the basis of identical cell populations measured by both approaches connects the two techniques. We develop a mathematical model to relate quantities exclusively observable by high-content single cell techniques to those measurable with high-content as well as high-throughput methods. The latter are defined as free variables, while the variables measurable with only one technique are described in dependence of those. It is the combination of data calibration and model into a single method that makes it possible to determine quantities only accessible by single cell assays but using high-throughput techniques. As an example, we apply our approach to the nucleocytoplasmic transport of STAT5B in eukaryotic cells.

Conclusions

The presented procedure can be generally applied to systems that allow for dividing observables into sets of free quantities, which are easily measurable, and variables dependent on those. Hence, it extends the information content of high-throughput methods by incorporating data from high-content measurements.

SUBMITTER: Pfeifer AC

PROVIDER: S-EPMC2928782 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Publications

Model-based extension of high-throughput to high-content data.

Pfeifer Andrea C AC Kaschek Daniel D Bachmann Julie J Klingmüller Ursula U Timmer Jens J

BMC systems biology 20100805

<h4>Background</h4>High-quality quantitative data is a major limitation in systems biology. The experimental data used in systems biology can be assigned to one of the following categories: assays yielding average data of a cell population, high-content single cell measurements and high-throughput techniques generating single cell data for large cell populations. For modeling purposes, a combination of data from different categories is highly desirable in order to increase the number of observab ...[more]

PMID: 20687942

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Project description:BackgroundDense genetic maps, together with the efficiency and accuracy of their construction, are integral to genetic studies and marker assisted selection for plant breeding. High-throughput multiplex markers that are robust and reproducible can contribute to both efficiency and accuracy. Multiplex markers are often dominant and so have low information content, this coupled with the pressure to find alternatives to radio-labelling, has led us to adapt the SSAP (sequence specific amplified polymorphism) marker method from a 33P labelling procedure to fluorescently tagged markers analysed from an automated ABI 3730 xl platform. This method is illustrated for multiplexed SSAP markers based on retrotransposon insertions of pea and is applicable for the rapid and efficient generation of markers from genomes where repetitive element sequence information is available for primer design. We cross-reference SSAP markers previously generated using the 33P manual PAGE system to fluorescent peaks, and use these high-throughput fluorescent SSAP markers for further genetic studies in Pisum.ResultsThe optimal conditions for the fluorescent-labelling method used a triplex set of primers in the PCR. These included a fluorescently labelled specific primer together with its unlabelled counterpart, plus an adapter-based primer with two bases of selection on the 3' end. The introduction of the unlabelled specific primer helped to optimise the fluorescent signal across the range of fragment sizes expected, and eliminated the need for extensive dilutions of PCR amplicons. The software (GeneMarker Version 1.6) used for the high-throughput data analysis provided an assessment of amplicon size in nucleotides, peak areas and fluorescence intensity in a table format, so providing additional information content for each marker. The method has been tested in a small-scale study with 12 pea accessions resulting in 467 polymorphic fluorescent SSAP markers of which 260 were identified as having been mapped previously using the radio-labelling technique. Heterozygous individuals from pea cultivar crosses were identifiable after peak area data analysis using the fluorescent SSAP method.ConclusionAs well as developing a rapid, and high-throughput marker method for genetic studies, the fluorescent SSAP system improved the accuracy of amplicon scoring, increased the available marker number, improved allele discrimination, and was sensitive enough to identify heterozygous loci in F1 and F2 progeny, indicating the potential to develop high-throughput codominant SSAPs.

Dataset Information

Model-based extension of high-throughput to high-content data.

Background

Results

Conclusions

Publications

Model-based extension of high-throughput to high-content data.

Similar Datasets

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