Characterization of Deletions using Oxford Nanopore Long-Read Sequencing

Abstract

Purpose : Deletions in Inherited Retinal Disease (IRD) genes are a common cause of IRDs, but they can be time consuming to characterise. Short-read next generation sequencing (NGS) can struggle to characterise deletions accurately, particularly in exome or targeted sequence, where it may detect a deletion but fail to capture the breakpoints. Genome sequencing allows more effective characterisation of deletions, but many deletions occur due to the presence of repetitive elements, which inhibit accurate alignment of short-read sequences. The ability to characterise deletions efficiently is essential for confirming and defining breakpoints detected by NGS. The goal of this study was to develop a cost-effective long-range sequencing method to characterise deletions.

Methods : Four cases with large deletions in IRD genes, detected in exome sequence but not further characterised, were selected. These were amplified with Long Range PCR primers encompassing the deletion, using the Sequel Prep Long-Range PCR kit, to give 9.5kb, 4.4kb, 5.8kb and 4.2kb products. Samples were purified with an AxyPrep Mag PCR Clean Up Kit. Sequencing was performed on a MinIon using separate Flongle Oxford Nanopore adaptors. 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 : This method allowed fast, cost-effective characterization of deletion breakpoints in EYS, PRPF31, CNGA1 (see figure) and CNGB1. A high output was achieved for each case, with maximum 1.5Gb. A high proportion of reads were on target, up to 99.78% of generated bases were in the target loci. For three of the cases, Nanopore sequencing captured the breakpoint at a nucleotide level. The presence of repetitive Alu elements at the breakpoints of the PRPF31 deletion prevented nucleotide level characterization. For all cases, Sanger sequencing was then performed to confirm the sequence of the breakpoint.

Conclusions : This study has designed and tested a long-range, low-cost, labour efficient strategy to characterise breakpoints in deletions. This will allow for rapid characterisation of deletions detected by NGS.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Characterized deletion in CNGA1. A) Coverage B) Flongle Reads C) Sanger Sequence verification. The top two traces are from the NA12878 control case. The bottom trace is from the deletion case. D) Gene context at the locus.