Introduction:
Recent studies in non-invasive biomarker research have demonstrated the potential of using cell-free nucleic acids isolated from blood plasma to serve as surrogates for solid tumors. Somatic mutations representing the tumors could be successfully detected from cell-free DNA (cfDNA) and cell-free RNA (cfRNA), providing new tumor assessment methods in addition to tissue biopsy. However, the low amount of circulating tumor fragments in the blood presents significant challenges for accurate variant detection with NGS assays. Moreover, utilization of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each blood sample.
Methods:
We demonstrate the feasibility for a comprehensive cell-free total nucleic acid NGS workflow that utilizes an extensive multi-gene panel to survey high-value variants relevant to multiple tumor types for liquid biopsy research. The method includes a novel sample preparation to simultaneously isolate cfDNA and cfRNA, followed by library preparation and sequencing on Ion Torrent™ platforms. 20 ng of input cell-free nucleic acid was used to prepare libraries. Multiple libraries were pooled together for templating on Ion Chef™ and sequenced on Ion S5™.
Results:
We have generated and streamlined an efficient sample-to-report NGS workflow using a broad single-pool multiplex assay panel to query more than 600 tumor driver and resistance hotspots with high sensitivity and specificity. The assay’s limit of detection at 0.1% allelic frequency enables accurate detection of low-abundance tumor variants from blood samples for liquid biopsy research. The broad content coverage of the panel encompasses hotspot SNVs and INDELs, CNVs, and gene fusions relevant to multiple tumor types, as well as expanded coverage of TP53 exon regions for TP53 mutation analysis. The different variant types were reliably detected in verified control samples harboring known variants, highlighting the capability of the assay to perform comprehensive mutation assessment. Sequencing on the Ion S5™ delivered > 95% on-target reads and uniform amplification with deep sequencing depth (> 40,000x).
Conclusions:
We demonstrate the ability to utilize isolated cell-free total nucleic acid for accurate detection of high-value variants implicated in multiple tumors Ion Torrent™ NGS. (For Research Use Only. Not for use in diagnostic procedures.)