Dataset Information

A droplet digital PCR detection method for rare L1 insertions in tumors.

ABSTRACT: BACKGROUND:The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. Through its mobility via a process called target primed reverse transcription (TPRT), L1 mobilization has resulted in over 100 de novo cases of human disease and has recently been associated with various cancer types. Large advances in high-throughput sequencing (HTS) technology have allowed for an increased understanding of the role of L1 in human cancer; however, researchers are still limited by the ability to validate potentially rare L1 insertion events detected by HTS that may occur in only a small fraction of tumor cells. Additionally, HTS detection of rare events varies greatly as a function of read depth, and new tools for de novo element discovery are needed to fill in gaps created by HTS. RESULTS:We have employed droplet digital PCR (ddPCR) to detect rare L1 loci in mosaic human genomes. Our assay allows for the detection of L1 insertions as rare as one cell in every 10,000. CONCLUSIONS:ddPCR represents a robust method to be used alongside HTS techniques for detecting, validating and quantitating rare L1 insertion events in tumors and other tissues.

SUBMITTER: White TB

PROVIDER: S-EPMC4297411 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A droplet digital PCR detection method for rare L1 insertions in tumors.

White Travis B TB McCoy Adam M AM Streva Vincent A VA Fenrich Joshua J Deininger Prescott L PL

Mobile DNA 20141231 1

<h4>Background</h4>The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. Through its mobility via a process called target primed reverse transcription (TPRT), L1 mobilization has resulted in over 100 de novo cases of human disease and has recently been associated with various cancer types. Large advances in high-throughput sequencing (HTS) technology have allowed for an increased understanding of the ro ...[more]

PMID: 25598847

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Project description:The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control. Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods. The single-laboratory validation study showed that performance requirements set for GMO analysis by real time PCR can also be used to assess dPCR-based methods. Moreover, trueness and precision were assessed for both simplex and duplex formats in a multi-laboratory validation study organised according to international standards. Overall, the data on trueness, repeatability and reproducibility precision resulting from the collaborative study are satisfying the acceptance criteria for the respective parameters as stipulated in the EU and other international guidance such as the Codex Committee on Methods of Analysis and Sampling (CCMAS). For instance, the duplex droplet dPCR method for MON810 showed relative repeatability standard deviations from 1.8% to 15.7%, while the relative reproducibility standard deviation was found to be between 2.1% and 16.5% over the dynamic range studied. Moreover, the relative bias of the dPCR methods was well below 25% across the entire dynamic range. In addition, other aspects supporting the application of digital PCR for the control of GMOs on the market were experimentally assessed such as the conversion of the measurement results from copy number ratio to mass fraction, the influence of the DNA extraction step and of the ingredient content. It was found that the DNA extraction step added only a limited contribution to the variability of the measurement results under the studied conditions. The decreasing amount of the target ingredient content may decrease the level of precision of the method, although within the acceptance range of GMO performance parameters. Highlights • In-house and collaborative validation studies of a droplet digital PCR method.• Trueness and precision assessed and compared for simplex and duplex formats in a collaborative study.• Evaluation of the influence of DNA extraction and ingredient content.• Description of compliance to EU and other international performance requirements for GMO analysis methods.• Application of conversion of measurement results from copy number ratios to mass fractions.

Dataset Information

A droplet digital PCR detection method for rare L1 insertions in tumors.

Publications

A droplet digital PCR detection method for rare L1 insertions in tumors.

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