Carmel Toomes

Background The widespread adoption of exome sequencing has greatly increased the rate of genetic diagnosis for inherited conditions. However, the detection and validation of large deletions remains challenging. While numerous bioinformatics approaches have been developed to detect deletions from whole ‐ exome sequencing and targeted panels, further work is typically required to define the physical breakpoints or integration sites. Accurate characterisation requires either expensive follow ‐ up whole ‐ genome sequencing or the time ‐ consuming, laborious process of PCR walking, both of which are challenging when dealing with the repeat sequences which frequently intersect deletion breakpoints. The aim of this study was to develop a cost‐effective, long‐range sequencing method to characterise deletions. Methods Genomic DNA was amplified with primers spanning the deletion using long‐range PCR and the products purified. Sequencing was performed on MinION flongle flowcells. The resulting fast5 files were basecalled using Guppy, trimmed using Porechop and aligned using Minimap2. Filtering was performed using NanoFilt. Nanopore sequencing results were verified by Sanger sequencing. Results Four cases with deletions detected following comparative read‐depth analysis of targeted short‐read sequencing were analysed. Nanopore sequencing defined breakpoints at the molecular level in all cases including homozygous breakpoints in EYS , CNGA1 and CNGB1 and a heterozygous deletion in PRPF31 . All breakpoints were verified by Sanger sequencing. Conclusions In this study, a quick, accurate and cost ‐ effective method is described to characterise deletions identified from exome, and similar data, using nanopore sequencing.