10 - Detection of Copy Number Variants by Next Generation Sequencing is Driven by Genomic DNA Integrity

Abstract

Copy number variants (CNVs) are common oncogenic drivers, impacting more of the cancer genome than all other types of mutations combined. Next generation sequencing (NGS) of cancer genomes is a highly sensitive method to detect CNVs from clinical sample types. However, as FFPE storage of clinical specimens severely damages DNA and results in poor sequencing coverage, detection of low-level CNVs (<3-fold) and CNVs in samples with low tumor cellularity remains challenging. Anchored Multiplex PCR (AMP™) is a target enrichment strategy that increases the read depth of target sequences by NGS. Molecular barcoded adapters ligated to DNA fragments prior to PCR enables amplification of fragments as small as 50 bp. AMP therefore increases read depth and coverage of target regions, thus enhancing the detection sensitivity of CNVs by downstream NGS assays. Since DNA damage from FFPE storage limits the number of molecules available for library preparation, the amount of amplifiable DNA fragments, rather than the total mass of DNA, drives library complexity, sequencing coverage, and detection sensitivity. To demonstrate the effect of input genomic DNA integrity on CNV detection sensitivity, we used the Archer PreSeq™ DNA QC Assay to determine the amounts of amplifiable genomes from over 150 FFPE tumor samples. AMP-based NGS using Archer VariantPlex assays in conjunction with Archer Analysis detected CNVs down to 2-fold and demonstrated that input genomic DNA integrity predicts the detection limit of CNVs. The accuracy of low-level CNV detection was also confirmed by serial dilution of CNV-positive samples.