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Practical guide for the comparison of two next-generation sequencing systems for solid tumour analysis in a universal healthcare system.

Author(s): Maxwell P, Hynes SO, Fuchs M, Craig S, McGready C, McLean F, McQuaid S, James J, Salto-Tellez M

Publication: J Clin Pathol, 2019, Vol. 72, Page 225-231

PubMed ID: 29386326 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared variant detection using different gene panels and investigated the reproducibility and analytical sensitivity of the Oncomine panel. The minimum DNA load of the Oncomine panel was also investigated. This study used residual FFPE lower gastrointestinal adenocarcinoma (n=17), lung adenocarcinoma (n=2), and malignant melanoma (n=9) specimens and a cell line. No details of specimen procurement, fixation, processing, or storage were provided. DNA was extracted using Maxwell 16, Roche Sample Prep Kit, or DNEasy kits following the manufacturer’s instructions (no information as to which kit for which specimen). NGS libraries were constructed using the Ampliseq Cancer Hotspot Panel v2, the Human Tumour Actionable Mutations Panel, and the Oncomine panel using 21 (Ampliseq and Actionable) or 19 (Oncomine) PCR cycles for initial target enrichment and seven (Ampliseq and Oncomine) or four PCR cycles for library amplification. Libraries were quantified by bioanalyzer. Templates for the Ampliseq and Oncomine panels were prepared using the OT2 200 Kit or the Hi-Q OT2 Kit and for the Actionable panel using MiSeq Reagent Kit v2. All data was processed using the same bioinformatics pipeline. Results were compared with reference data produced by quantitative PCR; Sanger sequencing of variants in IDH1 and 2, BRAF, KRAS, NRAS, and PIK3CA; and NGS analysis using the Ampliseq Cancer Hotspot Panel v2. The analytical sensitivity of the Oncomine panel was determined by dilution of variant samples into wildtype. The reproducibility of the Oncomine panel was tested by repeating the runs on the same chip and on separate chips. The effect of input on variant detection was evaluated by using various starting amounts of DNA starting with (between 0.2 ng and 34 ng).

   Summary of Findings:

   The percentage of amplicon targets with ≥500 X reads was slightly higher for the Actionable panel than the Oncomine panel (>99% versus 98.2). A total of 44, 146, and 199 variants were identified using the Actionable, Oncomine, and Hotspot panels, respectively. Of the 44 variants identified using the Actionable panel, 30 were also identified using the Oncomine and Hotshot panels, 11 were identified only with the Oncomine panel, and three were unique to the Actionable panel. Of the 146 variants identified using the Oncomine panel, 102 were also identified using the Cancer Hotspot panel and three were unique to the Oncomine panel. Results were strongly correlated between the Oncomine and the Hotspot panels (R=0.975, P<0.001), the Oncomine and the Actionable panels (R=0.977, P<0.001), and between the Actionable and Hotspot panels (R=0.974, P<0.001). Importantly, the Oncomine panel showed good reproducibility between separate chips and runs. The variant frequency declined and number of background variants decreased with decreasing input below 4 ng. The overall sensitivity of the Oncomine panel was 5%.

   Biospecimens

   • Tissue - Small Bowel
   • Tissue - Skin
   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Melanoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Next generation sequencing Specific	Template/input amount	34 ng 17 ng 9 ng 4 ng 2 ng 1 ng 0 ng Diluted to contain 5-30% mutant DNA
   Next generation sequencing Specific	Technology platform	Ampliseq Cancer Hotspot Panel v2 Human Tumour Actionable Mutations Panel Oncomine Panel

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