Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA

Florence Mauger,Caroline Horgues,Lilia Mesrob,Nouara Oussada,Jean-François Deleuze,Morgane Pierre-Jean

doi:10.1038/s41598-020-63102-8

Abstract

Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnosis, prognosis and monitoring treatment of disease. However, a sensitive and specific whole-genome sequencing (WGS) method is required to identify novel genetic variations (i.e., SNVs, CNVs and INDELS) on ccfDNA that can be used as clinical biomarkers. In this article, five WGS methods were compared: ThruPLEX Plasma-seq, QIAseq cfDNA All-in-One, NEXTFLEX Cell Free DNA-seq, Accel-NGS 2 S PCR FREE DNA and Accel-NGS 2 S PLUS DNA. The Accel PCR-free kit did not produce enough material for sequencing. The other kits had significant common number of SNVs, INDELs and CNVs and showed similar results for SNVs and CNVs. The detection of variants and genomic signatures depends more upon the type of plasma sample rather than the WGS method used. Accel detected several variants not observed by the other kits. ThruPLEX seemed to identify more low-abundant SNVs and SNV signatures were similar to signatures observed with the QIAseq kit. Accel and NEXTFLEX had similar CNV and SNV signatures. These results demonstrate the importance of establishing a standardized workflow for identifying non-invasive candidate biomarkers. Moreover, the combination of variants discovered in ccfDNA using WGS has the potential to identify enrichment pathways, while the analysis of signatures could identify new subgroups of patients.

Highlights

The analysis of circulating cell-free DNA from plasma bears great promise for diagnosis, prognosis and monitoring the treatment of cancer[1]
The extractions were performed using the commonly used QIAamp Circulating Nucleic Acid kit starting with 1 mL of plasma and using 100 μL of elution volume. Circulating cell-free DNA (ccfDNA) was quantified using Fluorometric assay and the fragment length sizes were analysed by electrophoresis to normalize each sample
Due to the lack of standard processing for ccfDNA, a workflow for sample preparation was performed to maximize the yield of ccfDNA including centrifugation, extraction, quantification, size analysis and normalization of samples

Summary

Introduction

The analysis of circulating cell-free DNA (ccfDNA) from plasma bears great promise for diagnosis, prognosis and monitoring the treatment of cancer[1]. Size analysis and quantification methods were used to evaluate the extracted ccfDNA. Sensitive approaches such as quantitative PCR, digital PCR, mass spectrometry and generation sequencing (NGS) are commonly applied to analyze extracted ccfDNA2. With the improvement of NGS analysis, whole-genome sequencing (WGS) is a great approach to identify all types of genomic alteration including single nucleotide variant (SNV), insertion and deletion (INDEL), copy number variation (CNV) and structural variant (SV) for the identification of candidate biomarkers in cancer[13]. Several specific and sensitive low-coverage sequencing approaches have been applied for the analysis of CNVs from cancer plasma samples[14,15,16,17,18,19,20].

Methods

Results

Discussion

Conclusion