Accurate and Automated Genotyping of the CFTR Poly-T/TG Tract with CFTR-TIPS

Cystic fibrosis is caused by biallelic pathogenic variants in the <i>CFTR</i> gene, which contains a polymorphic (TG)_mT_n sequence (the "poly-T/TG tract") in intron 9. While T₉ and T₇ alleles are benign, T₅ alleles with longer TG repeats, e.g., (TG)₁₂T₅ and (TG)₁₃T₅, are clinically significant. Thus, professional medical societies currently recommend reporting the TG repeat size when T₅ is detected. Sanger sequencing is a cost-effective method of genotyping the (TG)_mT_n tract; however, its polymorphic length substantially complicates data analysis. We developed <i>CFTR</i>-TIPS, a freely available web-based software tool that infers the (TG)_mT_n genotype from Sanger sequencing data. This tool detects the (TG)_mT_n tract in the chromatograms, quantifies goodness of fit with expected patterns, and visualizes the results in a graphical user interface. It is broadly compatible with any Sanger chromatogram that contains the (TG)_mT_n tract ± 15 bp. We evaluated <i>CFTR</i>-TIPS using 835 clinical samples previously analyzed in a CLIA-certified, CAP-accredited laboratory. When operated fully automatically, <i>CFTR</i>-TIPS achieved 99.8% concordance with our clinically validated manual workflow, while generally taking less than 10 s per sample. There were two discordant samples: one due to a co-occurring heterozygous duplication that confounded the tool and the other due to incomplete (TG)_mT_n tract detection in the reverse chromatogram. No clinically significant misclassifications were observed. <i>CFTR</i>-TIPS is a free, accurate, and rapid tool for CFTR (TG)_mT_n tract genotyping using cost-effective Sanger sequencing. This tool is suitable both for automated use and as an aid to manual review to enhance accuracy and reduce analysis time.