Dataset Information

PRIMEval: Optimization and screening of multiplex oligonucleotide assays.

ABSTRACT: The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and thus lower amplification efficiencies. We have developed a software workflow with three underlying algorithms that differ in their use case and specificity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method for the prediction of oligonucleotide hybridization events including resulting products and probes, self-dimers, cross-dimers and hairpins at different experimental conditions. The developed method allows explaining the observed artefacts through in silico WGS data and thermodynamic predictions. PRIMEval is available publicly at https://primeval.ait.ac.at.

SUBMITTER: Conzemius R

PROVIDER: S-EPMC6917790 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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PRIMEval: Optimization and screening of multiplex oligonucleotide assays.

Conzemius Rick R Hendling Michaela M Pabinger Stephan S Barišić Ivan I

Scientific reports 20191217 1

The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and thus lower amplification efficiencies. We have developed a software workflow with three underlying algorithms that differ in their use case and specificity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method ...[more]

PMID: 31848453

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Project description:Analysis of p53 binding sites using multiplex enhancer reporter assays, ChIP-seq data and RNA-seq data. Transcription factors establish and maintain the specific transcriptome of a cell by binding to genomic regulatory regions, thereby regulating the transcription of their target genes. Like many transcription factors, the DNA sequence-specific binding preferences of p53 are known. However, it remains largely unclear what distinguishes functional enhancers from other bound genomic regions that have no regulatory activity. In addition, the genome is scattered with seemingly perfect recognition sequences that remain unoccupied. To disentangle the rules of genome-wide p53 binding, we employed two complementary techniques of multiplex enhancer-reporter assays, one using barcoded reporters and the other using enhancer self-transcription. We compared the activity of more than one thousand candidate p53 enhancers under loss and gain of p53 conditions and identified several hundred high-confidence p53-responsive enhancers. Strikingly, the large majority (99%) of these target enhancers can be characterized and distinguished from negative sequences by the occurrence of a single p53 binding site. By training a machine learning classifier on these data, and integrating the resulting genome-wide predictions with fifteen publicly available human p53 ChIP-seq data sets, we identified a consensus set of 1148 functional p53 binding sites in the human genome. Unexpectedly, this direct p53 cistrome is invariably used between cell types and experimental conditions, while differences between experiments can be largely attributed to indirect non-functional binding. Our data suggest that direct p53 enhancers function in a context-independent manner and do not contain obvious combinatorial complexity of binding sites for multiple transcription factors. They represent a class of unsophisticated cell-autonomous enhancers with a single binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors. This suggests that context-dependent regulation of p53 target genes is not encoded in the p53 enhancer, but at different upstream or downstream layers of the cell's gene regulatory network.

Dataset Information

PRIMEval: Optimization and screening of multiplex oligonucleotide assays.

Publications

PRIMEval: Optimization and screening of multiplex oligonucleotide assays.

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