Efficient variant phasing utilizing a replication cycle reaction system

Purpose: When 2 heterozygous variants are detected in autosomal recessive disease genes, determining whether they are in cis or in trans is essential. Subcloning polymerase chain reaction products or complementary DNA is limited by variant distance (up to 10 kb) and complementary DNA availability. Droplet digital polymerase chain reaction, effective up to 100 kb, faces probe design challenges. We used replication cycle reaction (RCR), which replicates large DNA fragments based on E. coli chromosome replication, to phase widely spaced heterozygous variants.
Methods: Circular DNA molecules were formed by ligating CRISPR/Cas9-cleaved genomic fragments with an oriC-AmpR cassette, then amplified by RCR. Using a genomic DNA (gDNA) sample that is previously analyzed by long-read sequencing, we optimized reaction conditions (including gDNA to oriC-AmpR cassette ratios) and validated phasing accuracy via electrophoresis and Sanger sequencing. Finally, we applied this method to 7 patients harboring 2 heterozygous pathogenic variants (4.3-152 kb apart).
Results: RCR amplified genomic regions up to 104 kb. Lower gDNA-to-cassette ratios favored monoallelic amplification, enabling straightforward phasing, whereas higher ratios yielded biallelic products requiring transformation-based allele separation. For variants 152 kb apart, an intervening single-nucleotide variant enabled phased reconstruction. Ultimately, RCR confirmed compound heterozygosity in all 7 patients.
Conclusion: This method effectively phases multiple heterozygous variants across large genomic distances.