Analysis of clinically relevant large tandem repeats using nanopore sequencing

Variable number tandem repeats (VNTRs) remain among the most challenging regions of the human genome to characterize, due to their repetitive structure and high sequence variability. Short-read sequencing lacks the resolution to span these regions, and even long-read variant callers often fail to resolve true allelic variation due to alignment ambiguity and motif heterogeneity. We present a novel bioinformatics workflow for the analysis of VNTRs from nanopore sequencing data. To our knowledge, it is the first to offer fully automated variant detection, classification of loss-of-function (LoF) variants, and integrated quality control using nanopore sequencing technology. The pipeline separates reads into alleles, constructs reference-free consensus sequences, and aligns motif structures for visualization. LoF variants are identified and reported at their exact position within the repeat. The method was validated using PCR amplicons from reference genomes HG001–HG004 for two genes harboring VNTRs (ACAN and MUC1), as well as four clinical control samples containing known frameshift mutations in the MUC1 VNTR. We further demonstrate its applicability to whole-genome nanopore datasets. Using our pipeline, it is now possible to completely analyse and characterise VNTRs as well as detect disease-causing variants in a cost and time-efficient manner using amplicon-based nanopore sequencing.