# Detection of Copy‐Number Variations in CNS Tumours From Off‐Target Reads of Hybrid‐Capture Sequencing

## TL;DR

This study shows that off-target sequencing data from small DNA panels can detect copy-number variations in brain tumors, matching results from traditional array methods.

## Contribution

The study demonstrates that off-target reads from small hybrid-capture NGS panels can reliably detect clinically relevant CNVs in CNS tumors.

## Key findings

- Off-target reads from small NGS panels detected 95% concordance with methylation array CNV profiles.
- Focal amplifications and homozygous deletions were reliably identified using off-target reads.
- Meningiomas and pilocytic astrocytomas showed WHO-relevant CNV alterations detectable via this method.

## Abstract

Copy number variations (CNVs) play a central role in the classification, grading and prognostication of central nervous system (CNS) tumours. While genome‐wide methylation arrays are widely used for CNV profiling, next‐generation sequencing (NGS) panels are increasingly implemented in routine diagnostics. We hypothesised that off‐target sequencing reads from small hybrid‐capture panels not specifically designed for copy‐number detection can yield clinically actionable genome‐wide CNV profiles. We analysed 60 CNS tumour samples, including glioblastomas, oligodendrogliomas, ependymal tumours, medulloblastomas and choroid plexus tumours using a small‐scale custom hybrid‐capture panel (< 0,2 Mb) and compared CNV profiles inferred from sequencing reads to those obtained with methylation arrays. Additionally, 58 meningiomas and 6 pilocytic astrocytomas with BRAF fusions were profiled with the same NGS panel. Across 527 chromosomal arm‐level alterations, concordance between NGS‐ and methylation‐derived profiles was 95%. All 19 focal amplifications (e.g., EGFR, MDM4 and MYCN) and the majority of homozygous CDKN2A/B deletions were correctly detected. In meningiomas, genome‐wide CNV profiling from off‐target reads identified WHO‐relevant alterations, including CDKN2A/B deletions and 1p/22q co‐deletions, supporting molecular upgrading in 9/58 (16%) of histologically lower grade tumours. Focal copy‐number variations on Chr7q suggestive of BRAF fusions were observed in 5/6 fusion‐positive pilocytic astrocytomas. These findings demonstrate that off‐target reads from minimal targeted NGS panels can generate genome‐wide CNV profiles, comparable to methylation array data, without the need for additional assays or specialised probe designs.

Off‐target reads from small hybrid‐capture NGS panels can be leveraged to generate genome‐wide copy‐number profiles.Clinically relevant chromosomal arm‐level changes and focal amplifications can be reliably detected using off‐target sequencing reads.This approach can be incorporated into existing diagnostic workflows without requiring additional assays or specialised panel designs.

Off‐target reads from small hybrid‐capture NGS panels can be leveraged to generate genome‐wide copy‐number profiles.

Clinically relevant chromosomal arm‐level changes and focal amplifications can be reliably detected using off‐target sequencing reads.

This approach can be incorporated into existing diagnostic workflows without requiring additional assays or specialised panel designs.

Off‐target sequencing reads from small hybrid‐capture NGS panels can be leveraged to reconstruct genome‐wide copy number variation (CNV) profiles in CNS tumours. Across diverse tumour types, CNV profiles derived from off‐target reads show high concordance with methylation array data and reliably detect clinically relevant arm‐level alterations, homozygous deletions and even nontargeted focal amplifications. This strategy enables comprehensive molecular classification and prognostication without additional assays, maximising diagnostic yield from routine targeted sequencing.