Birgitte B Simen1, Lina Yin, Chirayu P Goswami, Kathleen O Davis, Renu Bajaj, Jerald Z Gong, Stephen C Peiper, Erica S Johnson, Zi-Xuan Wang. 1. From the Genomic Pathology Laboratory, Thomas Jefferson University Hospital (Dr Simen, Mr Goswami, and Ms Davis), Philadelphia, Pennsylvania; and the Departments of Pathology, Anatomy, & Cell Biology (Drs Yin, Bajaj, Gong, Peiper, Johnson, and Wang); and Surgery (Dr Wang), Thomas Jefferson University, Philadelphia. Drs Simen and Yin, Mr Goswami, and Ms Davis contributed equally to this article.
Abstract
CONTEXT: Next-generation sequencing allows for high-throughput processing and sensitive variant detection in multiple genes from small samples. For many diseases, including cancer, a comprehensive mutational profile of a targeted list of genes can be used to simultaneously inform patient care, establish eligibility for ongoing clinical trials, and further research. OBJECTIVE: To validate a pan-cancer, next-generation-sequencing assay for use in the clinical laboratory. DESIGN: DNA was extracted from 68 clinical specimens (formalin-fixed, paraffin-embedded; fine-needle aspirates; peripheral blood; or bone marrow) and 5 normal controls. Sixty-four DNA samples (94%; 64 of 68) were successfully processed with the TruSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) and sequenced in 4 sequencing runs. The data were analyzed at 4 different filter settings for sequencing coverage and variant frequency cutoff. RESULTS: Libraries created from 40 specimens could be successfully sequenced in a single run and still yield sufficient coverage for robust data analysis of individual samples. Sensitivity for mutation detection down to 5% was demonstrated using dilutions of clinical specimens and control samples. The test was highly repeatable and reproducible and showed 100% concordance with clinically validated Sanger sequencing results. Comparison to an alternate next-generation sequencing technology was performed by also processing 9 of the specimens with the AmpliSeq Cancer Hotspot Panel (version 2; Life Technologies, Grand Island, New York). Thirty of the 31 (97%) TruSeq-detected variants covered by the designs of both panels were confirmed. CONCLUSIONS: A sensitive, high-throughput, pan-cancer mutation panel for sequencing of cancer hot-spot mutations in 42 genes was validated for routine use in clinical testing.
CONTEXT: Next-generation sequencing allows for high-throughput processing and sensitive variant detection in multiple genes from small samples. For many diseases, including cancer, a comprehensive mutational profile of a targeted list of genes can be used to simultaneously inform patient care, establish eligibility for ongoing clinical trials, and further research. OBJECTIVE: To validate a pan-cancer, next-generation-sequencing assay for use in the clinical laboratory. DESIGN: DNA was extracted from 68 clinical specimens (formalin-fixed, paraffin-embedded; fine-needle aspirates; peripheral blood; or bone marrow) and 5 normal controls. Sixty-four DNA samples (94%; 64 of 68) were successfully processed with the TruSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) and sequenced in 4 sequencing runs. The data were analyzed at 4 different filter settings for sequencing coverage and variant frequency cutoff. RESULTS: Libraries created from 40 specimens could be successfully sequenced in a single run and still yield sufficient coverage for robust data analysis of individual samples. Sensitivity for mutation detection down to 5% was demonstrated using dilutions of clinical specimens and control samples. The test was highly repeatable and reproducible and showed 100% concordance with clinically validated Sanger sequencing results. Comparison to an alternate next-generation sequencing technology was performed by also processing 9 of the specimens with the AmpliSeq Cancer Hotspot Panel (version 2; Life Technologies, Grand Island, New York). Thirty of the 31 (97%) TruSeq-detected variants covered by the designs of both panels were confirmed. CONCLUSIONS: A sensitive, high-throughput, pan-cancer mutation panel for sequencing of cancer hot-spot mutations in 42 genes was validated for routine use in clinical testing.
Authors: Manuele G Muraro; Simone Muenst; Valentina Mele; Luca Quagliata; Giandomenica Iezzi; Alexandar Tzankov; Walter P Weber; Giulio C Spagnoli; Savas D Soysal Journal: Oncoimmunology Date: 2017-05-30 Impact factor: 8.110
Authors: M Giefing; M Wierzbicka; K Szyfter; J C Brenner; B J Braakhuis; R H Brakenhoff; C R Bradford; J A Sorensen; A Rinaldo; J P Rodrigo; R P Takes; A Ferlito Journal: Eur J Cancer Date: 2016-02-04 Impact factor: 9.162
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