TaKoHigh enables accurate variant calling and phasing of PCR-based long-read sequencing data

Yuka Eura; Atsushi Takahashi; Sheng Ye; Kazuya Sakai; Masanori Matsumoto; Koichi Kokame

doi:10.51094/jxiv.1517

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Yuka Eura Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center https://researchmap.jp/YMT180615
Atsushi Takahashi Department of Genomic Medicine, Omics Research Center, National Cerebral and Cardiovascular Center https://researchmap.jp/atstaka
Sheng Ye Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center
Kazuya Sakai Department of Blood Transfusion Medicine, Nara Medical University https://researchmap.jp/galapagos1986
Masanori Matsumoto Department of Hematology, Department of Blood Transfusion Medicine, Nara Medical University
Koichi Kokame Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center https://orcid.org/0000-0002-9654-6299 https://researchmap.jp/kkame

DOI:

https://doi.org/10.51094/jxiv.1517

キーワード:

long-read sequencing、 PCR amplicons、 variant calling、 haplotype phasing、 chimeric reads、 ADAMTS13、 thrombotic thrombocytopenic purpura

抄録

Long-read sequencing (LRS) is a powerful approach for analyzing causative variants in hereditary diseases. When the target gene is known, long-range PCR amplicons provide high coverage and efficiency. However, artifacts such as chimeric reads, allelic imbalance, and uneven coverage across overlapping amplicons can compromise the accuracy of variant calling and phasing. Yet no existing software is tailored to the properties of PCR-based LRS. We developed TaKoHigh, the first variant calling and phasing tool optimized for LRS of PCR amplicons. TaKoHigh analyzes each amplicon individually, applies thresholds based on allelic balance, and connects haplotypes through overlaps between adjacent amplicons. In ADAMTS13-associated cases, TaKoHigh achieved 98% variant calling accuracy, outperforming Clair3 (64%) and Longshot (50%). It also successfully resolved compound heterozygosity in cases where conventional tools failed. TaKoHigh enables robust interpretation of PCR-based LRS data without requiring specialized experimental protocols, making it broadly applicable in both clinical and research settings.

利益相反に関する開示

The authors declare no competing interests.

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