PO-086 An efficient ion torrent™ next generation sequencing workflow for liquid biopsy research to assess cell-free total nucleic acid

Abstract

IntroductionLiquid biopsy research contributes to precision medicine initiatives and enables minimally invasive and inexpensive sampling compared to traditional tissue biopsy. However, the low amount of circulating tumour nucleic acid fragments in the blood presents significant challenges for accurate variant detection using NGS technology. Utilisation of both cell free (cf) DNA and cf RNA requires methods capable of interrogating both types of analytes to maximise the utility of each blood sample. We described here a two days sample-to-report workflow using Oncomine Pan-Cancer Cell-Free Assay that surveys oncology variants across multiple tumour types and simultaneously detects single nucleotide variants (SNVs) and structural variants such as copy number variations (CNVs), gene fusions as well as exon skipping.Material and methodsCell Free Total Nucleic Acid (cfTNA) was extracted using MagMAX Cell-Free TNA Isolation Kit. Internal 0.1% or 0.5% cfDNA reference materials were used to evaluate SNV sensitivity and specificity. For CNV sensitivity and specificity evaluation, cfDNA from CNV positive cell lines were titrated into normal donor plasma cfTNA background. All controls were verified with orthogonal assays of dPCR. Libraries were manually prepared and sequenced with Ion Chef and S5 XL System. Data analysis was performed in Torrent SuiteTM 5.6 and Ion Reporter 5.6.Results and discussionsThe Pan-Cancer Cell Free Research Assay utilised a single-pool multiplex assay to query more than 900 tumour driver and resistance hotspots. The broad content panel encompasses SNVs, CNVs, fusions, exon skipping as well as expanded coverage of TP53 exon regions for TP53 mutation analysis. The entire work flow (sample-to-report) could be as less as 30 hours and is compatible with Oncomine knowledgebase that allows customers easy access to the variant reports. The limit of detection (LOD) of 0.5% and 0.1% allelic frequency can be achieved with sensitivity of >99% and 90% respectively, both specificity >99.9%. Detection as low as 1.3 fold gene amplification can be obtained for 12 CNV targets with sensitivity of >96% and specificity of >99%. For fusion detection, as low as 1% RNA fusions in cfTNA can be achieved.ConclusionThe Pan-Cancer Cell Free Research Assay provides an easy and quick NGS workflow that simultaneously analyses SNVs, CNVs, gene fusions and exon skipping across 52 genes associated multiple cancers. The 0.1% LOD enables accurate detection of low-abundance tumour variants for liquid biopsy research.