Efficient variant phasing utilizing a replication cycle reaction system

Abstract When two heterozygous variants are detected in genes for diseases with autosomal recessive inheritance, determining whether the two variants are located in cis or in trans is crucial. Subcloning long-range PCR products or cDNA is limited by factors such as the distance between variants (up to 10 kb) and cDNA availability. Droplet digital PCR, effective up to distances of 100 kb, faces challenges in specific probe design. We utilized replication cycle reaction (RCR) for amplifying large genomic DNA segments with multiple heterozygous variants. RCR is an in vitro replication cycle based on chromosome replication in Escherichia coli. Circular DNA molecules were generated by combining CRISPR/Cas9-cleaved genomic DNA fragments with an oriC–AmpR cassette, followed by amplification through RCR. Various molar ratios of gDNA to the oriC–AmpR cassette were evaluated to optimize the ligation step. We analyzed gDNAs from seven patients carrying two heterozygous pathogenic variants with distances ranging from 4.3 to 152 kb. A genomic region up to 104 kb could be amplified by RCR. A higher input of the oriC–AmpR cassette resulted in a higher rate of successful RCR amplification and a lower rate of successful monoallelic amplification. Monoallelic clonal amplification occurred in six patients, facilitating a rapid determination of variant phases. A haplotype was successfully reconstructed using an SNP located 78 kb away from the variant in one patient with two variants separated by a 152 kb distance. Our method proves particularly valuable for phasing multiple heterozygous variants separated over long genomic distances.