AmplideX® PCR/CE FMR1 Reporter


Introduction: In recent years, significant advancements in PCR technology have enabled the use of a PCR-only workflow for size analysis of the CGG-repeat region in the X-linked FMR1 gene. This genetic locus is directly associated to the transmission and manifestation of fragile X syndrome and a number of related disorders. To date, fragment size analysis of FMR1 PCR products is conducted with significant hands-on manipulation by trained operators, which can be laborious with larger sample sets.

We describe the development and implementation of a novel algorithm for fully automated and highly accurate analysis of the FMR1 CGG repeat region. This approach provides integrated QC checks, safeguards interpretative accuracy and consistency, and streamlines data analysis.

Methods: Fragment size analysis (.fsa) files or corresponding GeneMapper® tables (v4.1, Applied Biosystems®), for 273 genomic DNA samples were generated using the AmplideX® FMR1 PCR assay (Asuragen, Inc.), and used as input data for algorithm development. Multiple signal processing strategies were employed to enable separation of convoluted information from both the gene-specific and repeat-primed peak profiles produced by the assay. Software performance was then evaluated against a comprehensive set of 1000 previously annotated clinical samples, which spanned normal to full-mutation FMR1 genotypes.

AmplideX Reporter Methods figure

Results: Software was designed to allow single-click analysis without the need to select electropherogram peaks for FMR1-related peaks or size standards. A comprehensive set of auto-tuning and quality control steps, including the use of AmplideX® FMR1 Control DNA, was embedded into the process to minimize error and increase reliability and robustness of performance across multiple platforms and input variables. Automated sizing of the FMR1 CGG repeat region was 100% concordant (within ±1 repeat) with corresponding manually-analyzed data across a 1000-member test sample set; results were obtained in less than 1% of the time needed for manual analysis. In addition, the software was able to identify the presence or absence of non-convoluted AGG interruptions in samples bearing alleles within the clinically-relevant risk range, in accord with known sample annotations. Finally, proof-of-concept was demonstrated for direct analysis of capillary electrophoresis raw data without an external ladder for size calibration.

AmplideX Reporter Screen Shot AAmplideX Reporter Screen Shot B







Screenshots of the application interface. A) Project submission B) Results visualization

Conclusions: Results from this study demonstrate the accurate and robust performance of a fully automated FMR1 analysis tool. These results underscore the potential utility of software integration into a comprehensive FMR1 PCR workflow that augments data quality, enhances interpretive reliability, and reduces analysis and reporting bottlenecks, particularly for high-volume testing labs.

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