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 analyze and characterize VNTRs as well as detect disease-causing variants in a cost and time-efficient manner using amplicon-based nanopore sequencing.

Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant form of muscular dystrophy caused by genetic or epigenetic changes within the D4Z4-r

Polyomaviruses have the potential to cause significant morbidity not only in transplant medicine, but also in other forms of disease or variants of immunosuppression. In kidney transplant recipients or recipients of human stem cell transplants, the BK-Virus is the major proponent of manifestations such as BKPyV-associated nephropathy or hemorrhagic cystitis. As no polyomavirus-specific drug with proven in vivo effects has been developed so far, methods to screen for such drugs are important. This work describes the establishment of a virus-secreting cell line. By infecting a pre-established monkey kidney cell line (COS-1) with a non-rearranged human BK polyomavirus isolated from a kidney transplant patient suffering from BKPyV-associated nephropathy, a continuously replicating cell type with consistent virus secretion could be established and was termed COSSA. Measurements of BKPyV replication, virion production, and secretion were performed both intracellularly and in the cell supernatant. Viral proteins such as VP1 and LTAg were accurately tracked by confocal microscopy, as well as by immunoblot and qPCR. An intracellular flow cytometry (FACS) assay detecting VP1 protein was established and revealed an expanded range of positive intracellular signals. The viruses produced proved to be infectious in human tubular epithelial cell lines. Long-range sequencing of the COSSA genome using Oxford Nanopore Technology revealed a total of five distinct BKPyV integration events. One integration of a partial BKPyV genome was located upstream of the epidermal growth factor receptor gene. The second and third, both truncated forms of integration, were close to histocompatibility gene locuses, while the fourth was characterized by a ninefold and the fifth by a fourfold tandem repeat of the BKPyV genome. From both of the repeat forms, virus replicates were derived showing deletions/duplications on early and late genes and inversions within the non-coding control region (NCCR). This pattern of repetitive viral genome integration is a potential key driver of enhanced viral replication and increased virion assembly, ultimately supporting efficient virus egress. Quantitative PCR analysis confirmed the release of approximately 10⁸/mL viral units per 48 h from 2 × 10⁵ COSSA cells into the culture supernatant. Notably, the NCCR region of the most frequent copies of circular virus and the integrated tetrameric tandem repeat exhibited a rearranged configuration, which may contribute to the observed high replication dynamics. The establishment of a consistent methodology to generate and secrete BKPyV from a cell line is expected to significantly facilitate antiviral drug development.

A bstract: Methods for sequence error correction can improve sequence accuracy; however, there can be unintended errors added during error correction. One such example is phase switching, whereby sequences derived from genomes containing more than one parental copy have contributions from more than one parental haplotype. Such switches are mistakes that can confound downstream analyses especially de novo genome assembly. While DNA sequences do not possess words such as linguistic languages, one can partition a DNA sequence into word-like objects called k-mers. K-mers include pieces of DNA sequence of k length that can describe various properties of DNA sequences. With regard to phase switching, there are k-mers present in one parental haplotype not found in other(s). These so-called hapmers can represent inaccuracies in that parental haplotype’s assembly but also correct, unique DNA variation. Here we investigated the effect of DNA sequence error correction on phase switching at the sequence/read level. Using several error-correction methods, we find all methods tested are similar to raw, presumably, phase-switch-free Oxford Nanopore Technologies (ONT) sequences in the percentage of readmers (k-mers from the ONT sequences) matching one parental haplotype’s hapmers. This work demonstrates an efficient method to assess if an error-correction method has introduced phase switching implemented in the Julia programming language.