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Introduction: Accurate characterization of variants in medically challenging genomic regions-including those with segmental duplications, pseudogenes, low-complexity sequences, and other repeat-rich loci-remains a major limitation of short-read sequencing.These regions contribute disproportionately to clinically relevant structural variation yet are often inaccessible to traditional sequencing pipelines because of mapping ambiguity and limited fragment length.Long-read platforms such as PacBio HiFi and Oxford Nanopore improve resolution but require dedicated instruments and high-molecular-weight DNA, often necessitating additional blood collection and specialized extraction workflow.Constellation Mapped Read Technology offers an alternative by enabling long-range genomic analysis on the standard Illumina NovaSeq X using routine clinical DNA preparations, providing a promising path toward broader adoption of long-read-level analysis.We evaluated its performance across a cohort specifically selected to include diverse and analytically difficult variant types encountered in clinical practice.Methods: Eight samples were included, all extracted using DNA preparation methods typical of short-read workflows, including both manual and automated protocols.DNA quality and storage history reflected real-world conditions, ranging from freshly isolated gDNA to samples stored at -20 C for two months, one year, two years, four years, and up to six years.The cohort was constructed to represent a broad spectrum of diagnostic challenges.HG002 (NA24385, GIAB) was included to benchmark performance against the CMRG_v1.00dataset, which provides long-read-derived truth sets for Challenging Medically-Relevant Genes.The remaining seven clinical samples had prior characterization by short-read ES/GS, long-range PCR, breakpoint PCR, karyotyping, or optical genome mapping.Targeted variants included an FXN GAA expansion (50-157 repeats), a t(5;17) translocation, a PMS2 exon 11-12 tandem duplication affected by PMS2CL interference, the MSH2 Boland inversion spanning exons 1-7, a complex inverted duplication on chr5q32.2, a large duplication on chr5q32.2involving LMNB1 with orientation-dependent clinical relevance, and two overlapping chr16 duplications resulting in four copies of TBX6 with unresolved phasing.Variant calls generated by Constellation were evaluated for breakpoint precision, orientation, phasing, and consistency with orthogonal results.Results: All samples produced usable data, demonstrating compatibility with a range of DNA qualities and extraction methods.Six of eight samples met optimal DNA QC (>70% fragments >10 kb), while one fibroblast-derived manually extracted sample and one sample stored for nearly six years showed reduced metrics yet remained interpretable.Against the Medically Challenging Genomic Regions benchmark, Constellation showed improved performance over short-read sequencing, increasing precision from 98.36% to 99.51% and sensitivity from 97.47% to 99.05% across all variants.Constellation characterized the FXN GAA repeat expansion and captured the full extent of the expanded alleles.The t(5;17) translocation was identified with breakpoint support consistent with the SV_BND pattern observed in short-read data, demonstrating that Constellation can surface balanced rearrangements even without prior knowledge.The PMS2 exon 11-12 duplication was resolved with basepair-level breakpoint precision, overcoming the challenges posed by PMS2/PMS2CL homology.The MSH2 Boland inversion was correctly detected, with attenuated signal corresponding to reduced DNA quality.The chr5q32.2 duplication involving LMNB1 was resolved as a direct tandem duplication using the colocation signal, which also revealed an SVA retrotransposon insertion at the breakpoint that prevented short-read mapping from determining orientation.Although GS detected the copy-number gain, only Constellation was able to define the breakpoint structure and its clinically relevant configuration.Conclusion: Constellation Mapped Read Technology demonstrated robust performance in medically challenging genomic regions and reliably resolved complex structural variants using standard clinical DNA inputs, including samples with reduced integrity.The platform consistently identified tandem duplications, repeat expansions, inversions, and other clinically important events.These findings support Constellation as a practical and scalable long-range sequencing solution suitable for integration into existing Illumina-based clinical workflows.