DEEPGENTM—A Novel Variant Calling Assay for Low Frequency Variants

Bernd Timo Hermann,Johannes Bhakdi,Samuel Schaible,Monika Elisabeth Hagen,Sebastian Pfeil,Nicole Groenke,Frédéric Ris,Robert Kunze

doi:10.3390/genes12040507

Abstract

Detection of genetic variants in clinically relevant genomic hot-spot regions has become a promising application of next-generation sequencing technology in precision oncology. Effective personalized diagnostics requires the detection of variants with often very low frequencies. This can be achieved by targeted, short-read sequencing that provides high sequencing depths. However, rare genetic variants can contain crucial information for early cancer detection and subsequent treatment success, an inevitable level of background noise usually limits the accuracy of low frequency variant calling assays. To address this challenge, we developed DEEPGENTM, a variant calling assay intended for the detection of low frequency variants within liquid biopsy samples. We processed reference samples with validated mutations of known frequencies (0%–0.5%) to determine DEEPGENTM’s performance and minimal input requirements. Our findings confirm DEEPGENTM’s effectiveness in discriminating between signal and noise down to 0.09% variant allele frequency and an LOD(90) at 0.18%. A superior sensitivity was also confirmed by orthogonal comparison to a commercially available liquid biopsy-based assay for cancer detection.

Highlights

Generation sequencing (NGS) has become an essential technology for an array of biological and medical disciplines [1]
Next generation sequencing (NGS) libraries were prepared from cfDNA according to the manufacturer’s instructions (Protocol based on QIAseq Targeted deoxyribonucleic acid (DNA) Panel Handbook (R2; May 2017), Qiagen, Hilden, Germany)
After unique molecular identifier sequence (UMI) attachment, target enrichment of ligated cfDNA was performed by PCR using target specific DEEPGENTM primers

Summary

Introduction

Generation sequencing (NGS) has become an essential technology for an array of biological and medical disciplines [1]. Detection of somatic variants provides a medically useful application of NGS technology to characterize changes at clinically relevant loci within a patient’s genome. The detection of single- and poly nucleotide variants within deoxyribonucleic acid (DNA) sequences facilitates advanced medical tasks such as diagnosing particular diseases, hereditary risk assessment, longitudinal evaluation of treatment effectiveness and gaining a deeper understanding of diseases [3,4,5]. To perform exactly these tasks with maximum efficiency, we developed DEEPGENTM, a novel variant calling assay which utilizes targeted, paired-end sequencing of short reads (Figure 1). The entire assay, including the bioinformatics pipeline, were optimized to detect an extensive set of oncology-relevant variants at very low allele frequencies from liquid biopsy-derived circulating tumour DNA (ctDNA)

Methods

Results

Conclusion