Analysis of circulating cell-free DNA (ccfDNA) from blood in the fields of life science, clinical research and beyond is expanding and has become widely accepted. The complete workflow entails collection and stabilization of blood, transport, plasma processing, extraction of ccfDNA and downstream analysis. Considering the usual low sample input and required high sensitivity for quality control (QC) and variant detection, optimal outcomes require verification of the entire workflow.
In this verification study, human blood was collected, stabilized, and then processed to isolate ccfDNA from plasma using the PAXgene Blood ccfDNA System for subsequent analysis by next-generation sequencing (NGS). To verify the workflow, several sample QC criteria were assessed at each step of the workflow, including ccfDNA yield, target enrichment, library preparation, target sequencing and analysis.
Blood from 60 healthy consented donors was collected into PAXgene Blood ccfDNA Tubes and stored for 7 days at room temperature (15–25°C) to simulate a typical processing delay in a routine setting. Automated ccfDNA extraction was performed on the QIAsymphony SP instrument using the QIAsymphony PAXgene Blood ccfDNA Kit and protocol. ccfDNA stability after blood storage was confirmed by qPCR. ccfDNA samples were sequenced on the QIAGEN GeneReader NGS System, including the GeneRead QIAact Actionable Insights Tumor (AIT) Panel for PCR target enrichment, library preparation on the QIAcube instrument, QC with capillary electrophoresis, sequencing on the GeneReader instrument and data management with the QIAGEN Clinical Insight (QCI) Analyze tool.
After blood storage for 7 days, ccfDNA yield was similar to ccfDNA yield observed directly after blood draw. All samples (60/60) passed the required QC criteria after target enrichment (amplicon size around 160 bp) and library preparation (amplicon size around 252 bp and absence of unspecific products <170 bp). Also, all GeneReader NGS acceptance criteria that defined “passed” or “out-of-spec” samples were passed after sequencing, including reads above average quality 25 with 90.16 ± 2.04% (acceptance criteria >80%), region of interest with coverage of bases >500x with 99.83 ± 0.47% (>90%) and region of interest with coverage of bases >200x with 99.99 ± 0.03% (>95%). Across all samples, 17 ± 5 different variants in 7 ± 1 different genes were identified with the AIT targeted panel.
NGS is one of the most important applications for analysis of ccfDNA in research and clinical settings. This study verified that ccfDNA stabilized and extracted with the PAXgene Blood ccfDNA System is highly suitable for NGS applications, meeting quality control acceptance criteria for 100% of analyzed samples.
Tomasz Krenz1, Andrea Ullius1, Ricardo Huebel1, Thorsten Voss1, Daniel Groelz1, Eric Provencher2, Timothy R. Buirkle2. 1 PreAnalytiX GmbH, Hilden, Germany; 2 BD, Franklin Lakes, NJ