475P - Optimising genomic testing for patients with central nervous system (CNS) tumours using oxford nanopore technology

Background

Oxford Nanopore Technologies (ONT) sequencing platform offers substantial advantages for identifying clinically relevant variants in tumour genomes. The longer read length of ONT data improves mapping in traditionally difficult regions of the genome such as repetitive sequences and allows phasing of breakpoints for complex structural variants (SVs). ONT sequencing can also identify methylated DNA base pairs, giving insight into the epigenetic changes occurring in tumour cells such as methylation of MGMT promoter and enabling diagnostic classification of CNS tumour subtypes. Therefore, there is an opportunity to use ONT sequencing for methylation-based analyses alongside genome-wide variant calling.

Methods

As a part of developing analytical pipeline for pilot study assessing clinical benefits of ONT-based Whole Genome Sequencing (WGS) we have benchmarked several tools for diagnostic classification against EPIC array data using a dataset of paediatric brain tumours and developed an in-house method to identify patterns of MGMT promoter methylation. We have also optimised available variant calling tools and assessed their quality against variants in clinically relevant genes derived from Illumina WGS data.

Results

Our analysis demonstrates that ONT-based classifiers are as accurate as the Heidelberg classifier using EPIC arrays. Assessment of MGMT promoter methylation at the read level provides insight into both the degree of methylation across the promoter region with resolution of individual CpGs and the frequency of methylated reads, which can be used to determine methylation zygosity, unlike existing MGMT promoter assays. We also present assessment of accuracy for variant calling (small variants, structural variants and copy number aberations) from ONT WGS data as well as examples of newly identified SVs in the regions of low complexity that were missed by Illumina WGS.

Conclusions

These developments represent significant progress towards implementing an ONT platform to maximise clinical benefits of WGS for patients with CNS tumours. These benefits are evident in the improved ability to call complex structural variants and to provide epigenomic information in a single test along with variant calling.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Genomics England.

Funding

Department of Health.

Disclosure

All authors have declared no conflicts of interest.