Haplotyping Using Long-Range PCR and Nanopore Sequencing of Phase Variants; Lessons Learned From the ABCA4 Locus

Short-read next-generation sequencing has revolutionized our ability to identify variants underlying inherited diseases, but it does not allow the phasing of variants to clarify their diagnostic interpretation. The advent of widespread, increasingly accurate long-read sequencing has opened up new applications not currently available through short-read next-generation sequencing. One such use is the ability to phase variants to clarify their diagnostic interpretation and to investigate the increasingly prevalent role of cis-acting variants in the pathogenesis of the inherited disease, so-called complex alleles. Complex alleles are becoming an increasingly prevalent part of the study of genes associated with inherited disease, for example, in ABCA4-related disease. We sought to establish a cost-effective method to phase contiguous segments of the 130kb ABCA4 locus by long-read sequencing of overlapping amplification products. Using the comprehensively characterized CEPH sample, NA12878, we verified the accuracy and robustness of our assay. However, in-field assessment of its utility using clinical test cases was hampered by the paucity and distribution of identified variants and by PCR chimerism, particularly where the number of PCR cycles were high. Despite this, we were able to construct robust phase blocks of up to 94.9kb, representing 73% of the ABCA4 locus. We conclude that, while haplotype analysis of variants located within discrete amplification products was robust and informative, the stitching-together of larger phase blocks using overlapping single-molecule reads remained practically challenging.