Automated Fragile X Analysis: Calculation of Triplet CGG Repeats & Percent Methylation

Fragile X Syndrome (FXS) is caused by expansions of a CGG triplet repeat in the FMR1 gene on the X chromosome, similar to trinucleotide expansions associated with other diseases such as Huntington's disease and myotonic dystrophy. In studies of FXS, the number of repeats and methylation status of the gene are associated with a constellation of disorders that impact a broad range of ages and populations. Advances in PCR-methods allow researchers to directly determine repeat length and methylation status using a two-color PCR approach.

GeneMarker® software is compatible with data from Asuragen®-AmplideX® or custom chemistries, output files from all major capillary electrophoresis instruments and Windows® 7-10, for rapid and accurate analysis of large volumes of single-dye or two-dye data associated with FXS. The linked Fragile X application automatically coordinates edits in Control and Digestion channels for two-dye projects and performs repetitive calculations, converting fragment size to repeat length and calculating percent methylation. Analysis parameters are easily customized to fit the SOP for the laboratory, with automated calculation of Correction Factors using a control sample with fragments of known CGG repeat length or a .txt file with the CGG repeat and bp size. Reporting options include .xls, .txt files of result tables, image files of individual sample reports, and color-coded project summary reports.

Review figures 1 – 4 below for an overview of the Fragile X analysis settings and reports

  • Reduction in analysis time by up to 50%
  • Reduction in errors, no data transfer
  • Accurate Fragile X Analysis
  • Single & Multi-color Analysis capabilities

Example of a Fragile X CGG repeat report

Figure 1: Final sample report from a single dye chemistry to calculate the number of CGG repeats for each fragment, including a similar Header, Conclusion/Authorization box, Electropherogram and Report Table with the calculated number of triplet repeats, size in bp, and peak height for each fragment.

Example of a Fragile X Methylation Report

Figure 2: Final sample report from two-dye methylation chemistry includes: Header with details from user management and analysis parameters, Conclusion/Authorization box, Electropherogram and Report table The report table contains the sample metrics for the digestion control and reference fragment, the calculated number of triplet repeats, size of fragment in base pairs, peak height, and calculated percent methylation for each fragment.

Specify the Fragile X settings appropriate for your laboratory

Figure 3: Parameters for Fragile X analysis are easily customized for single-dye or two-dye projects. Control (calibrator) samples with known repeat sizes can be selected from the drop-down menu, or a .txt file can be imported with the expected repeat and bp size for the application. Co and Mo factors are automatically calculated when the control sample is selected.

Example Project Summary Report

Figure 4: Project Summary report example; color coded for fragments that did not meet specified parameters.

Application Notes:


MLPA® is the registered trademark of MRC Holland