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Abstract Amplifying long and complex genomic regions with high fidelity is essential for accurate long-read sequencing, especially in diseases linked to repeat expansions, where polymerase slippage and stalling can compromise sequencing results, ultimately impacting disease diagnosis and prognosis. In Fragile X Syndrome, for example, amplifying the FMR1 gene is challenging due to CGG repeat expansions that often exceed 200 repeats. Also, Short-read Next Generation Sequencing struggles to detect these expansions accurately, relying on computational algorithms for sequence assembly, this can reduce sequence and, therefore, diagnostic accuracy. Consequently, long-read sequencing offers a more reliable method for directly identifying longer sequences, with the amplification step being crucial to ensuring successful detection. We evaluated the performance of DeCodi-Fi, a novel high-fidelity polymerase, by amplifying and sequencing genes with tandem repeats linked with the following disease-associated genes: FMR1 (Fragile X Syndrome), C9orf72 (Amyotrophic Lateral Sclerosis, ALS), IT15 (Huntington*s Disease), and X25 (Friedreich*s Ataxia), as well as long templates exceeding 12 kb, including BRCA1 (Breast Cancer) and SMN1 (Spinal Muscular Atrophy) in previously reported healthy and diagnosed individuals. Sequencing analysis was performed to assess DeCodi-Fi*s accuracy and ability to amplify complex templates without introducing errors. Its performance was also compared against other commercial high-fidelity polymerase. We successfully optimized PCR conditions for four repeat expansion targets and two long amplicons of clinical relevance. Long-read sequencing libraries were generated and analyzed using Oxford Nanopore technology. Consensus sequences were mapped to curated data from the corresponding Coriell samples. DeCodi-Fi polymerase successfully amplified repeat expansions with +/- 1 repeat accuracy. For the non-repeated long amplicons we obtained full coverage and polymerase error rates below the sequencing error threshold, when compared to the repository sequence data. This study demonstrates that DeCodi-Fi is capable of amplifying complex and long DNA templates with minimal errors, providing a reliable tool for long-read sequencing involving challenging genomic regions.