PB0305 - New program optimized for long-read sequencing of targeted gene PCR products

Background: Homozygous or compound heterozygous pathogenic variants in the ADAMTS13 gene, which encodes a von Willebrand factor-cleaving protease, can cause congenital thrombotic thrombocytopenic purpura (cTTP). We previously analyzed the ADAMTS13 gene exons by conventional Sanger sequencing in approximately 60 Japanese patients with suspected cTTP and identified causative variants in both alleles in almost all cases, but not in the remaining four cases.

Aims: We attempted to establish a highly accurate long-read sequencing (LRS) method to identify the latent causative variants.

Methods: Six to eight overlapping products (10-15 kb) of long-range PCR were designed and obtained to cover the entire ADAMTS13 gene (45 kb). All PCR products were subjected to library preparation and LRS using an Oxford Nanopore system.

Results: Haplotype blocks were generated using existing programs (Longshot, Clair3, etc.) and candidate variants with phasing information were identified. However, validation with Sanger sequencing revealed that some variants were missed or had incorrect haplotype information. Therefore, we developed a new program optimized for LRS of overlapping PCR products, and named it TaKoHigh (Targeted Amplicon-based valiant Calling and phasing Optimized for High-throughput long read sequencing). Throughly validation using Sanger sequencing of the ADAMTS13 gene of one patient showed that the phasing by TaKoHigh, Longshot, and Clair3 were correct, incorrect, and incorrect, respectively, and that the variant detection rates were 98% (TaKoHigh), 50% (Longshot), and 63% (Clair3).

Conclusion(s): Long-range PCR occasionally produces unexpected artifacts such as allelic chimera and unbalanced amplification between alleles, which interfere with correct phasing and variant calling in the existing programs. Our newly developed program, TaKoHigh, overcame the misphasing and miscalling and led to more reliable results.