title: Validation of a next-generation-sequencing cancer panel for use in the clinical laboratory.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.affiliation: 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.
authors.forename: Birgitte B
authors.initials: BB
authors.lastname: Simen
authors.forename: Lina
authors.initials: L
authors.lastname: Yin
authors.forename: Chirayu P
authors.initials: CP
authors.lastname: Goswami
authors.forename: Kathleen O
authors.initials: KO
authors.lastname: Davis
authors.forename: Renu
authors.initials: R
authors.lastname: Bajaj
authors.forename: Jerald Z
authors.initials: JZ
authors.lastname: Gong
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Peiper
authors.forename: Erica S
authors.initials: ES
authors.lastname: Johnson
authors.forename: Zi-Xuan
authors.initials: ZX
authors.lastname: Wang
mesh_terms: D019411:Clinical Laboratory Techniques / Q000379:methods*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020022:Genetic Predisposition to Disease / Q000235:genetics; D005820:Genetic Testing / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D004273:DNA, Neoplasm