20 research outputs found
Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.
Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications
Reflex™: a novel method to sequence barcoded long-range PCR products in a pooled population of hundreds of DNA samples
Reflex™: a novel method to sequence barcoded long-range PCR products in a pooled population of hundreds of DNA samples
Reflex: intramolecular barcoding of long-range PCR products for sequencing multiple pooled DNAs
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Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.
Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications
Amplification sensitivity analysis for <i>FGFR1</i>, <i>EGFR</i>, <i>ERBB2</i> and <i>MET</i>.
<p>Amplification sensitivity analysis for <i>FGFR1</i>, <i>EGFR</i>, <i>ERBB2</i> and <i>MET</i>.</p
Sensitivity for SNVs (A, C) and indels (B, D).
<p><b>A</b> and <b>B</b> show the sensitivity as a function of the allele fraction of the reference mutations. Each line represents a different operator/Lot combination. <b>C</b> and <b>D</b> show the full set of calls for all combinations of dilution/variant (vertical) and repeat/operator/lot (horizontal). Blue rectangles represent mutations that were detected and grey represents those missed. Panel E shows for SNVs the estimated allele fraction compared between InVisionFirst (blue box-plots) and the reference as estimated by Horizon using ddPCR (red crosses).</p
Fusion sensitivity analysis.
<p>Blue rectangles represent fusions that were detected and grey represents those missed. (A) Dilution of Horizon reference material containing 2 fusions (ALK and ROS1) across dilution levels (vertical) and operator/lot (horizontal), (B) Set 1 of contrived material based on published DNA breakpoints (AF 1% and 0.5%, 2 operators), (C) Set 2 of contrived material based on published DNA breakpoints (AF 1% and 0.5%, 2 operators, 2 reagent lots) and (D) Contrived material based on randomly generated fusion breakpoints. Different operators performed different parts of this fusion study.</p