ABCA7 VNTR characterization based on raw current PromethION sequencing data. ABCA7 VNTR characterization with PromethION

OBJECTIVES We recently identified strong association between expanded alleles of an ABCA7 VNTR and Alzheimer’s disease (AD). Analysis of this tandem repeat (TR), however, is only possible through low-throughput Southern blotting, which precludes further characterization and implementation in the clinic. We aimed to provide a high-throughput long-read sequencing alternative to assess the length, sequence and methylation state of the ABCA7 VNTR. METHODS We performed whole genome long-read sequencing on DNA of eleven patients with AD or frontotemporal lobar dementia and healthy control individuals using the recently released PromethION® sequencing platform (Oxford Nanopore Technologies®). We subsequently characterized the ABCA7 VNTR, by developing a new pattern recognition algorithm based on dynamic time warping of raw long-read sequencing data. We validated the results with Southern blotting. RESULTS With a single sequencing run per individual, we were able to detect all VNTR alleles, which ranged from 300 to more than 10000 bases in length. We obtained length estimates with more than 90% accuracy and high precision (5.6% relative standard deviation). We consistently identified alternative TR sequence motifs, allowing distinction of VNTR alleles with homozygous length. Furthermore, our approach capacitates the detection of nucleotide modifications (e.g. methylation) within the TR. CONCLUSIONS We provide a new long-read sequencing based method to study the ABCA7 VNTR at an unprecedented resolution, enabling a better understanding of the disease-associated mechanisms. In addition, our approach opens the possibility for whole-genome analysis of (expanded) TRs which will lead to identification of novel disease causing variants and improved diagnostics.