Comprehensive evaluation and validation of targeted next-generation sequencing performance in two clinical laboratories.
Next-generation sequencing (NGS) has led to breakthroughs for genetic and genomic analyses and personalized medicine approaches for many diseases. More and more clinical laboratories are using NGS as a genetic screening tool for providing mutation information that is used to select the best treatment regimens for cancer patients. However, several obstacles prevent the routine implementation of NGS technology into the clinical molecular diagnosis setting: the sophisticated sample preparation process, high cost, time-consuming data analyses, as well as the reproducibility and accuracy of interpretation. To systematically evaluate the performance and quality of targeted NGS cancer panel analyses in clinical laboratories, we performed three different tests: i) laboratory-to-laboratory accuracy test, ii) intra-laboratory precision validation, and iii) limit of detection test, using formalin-fixed, paraffin-embedded cancer tissue specimens, cell lines and mutation positive DNA. A laboratory-to-laboratory accuracy test performed using 51 samples showed 100% sensitivity and 99.97% specificity. For the intra-laboratory precision test, 100% reproducibility was observed. For the limit of detection test, KRAS mutations from samples diluted from 70 to 2% of mutant allele frequencies were detected correctly. We believe that the present study demonstrated the feasibility of clinical implementation of a targeted NGS cancer panel analysis for personalized medicine.
Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
Pedro
P
Mendez
CureSeq Inc., Brisbane, CA 94005, USA.
Jennifer
J
Dang
CureSeq Inc., Brisbane, CA 94005, USA.
James Wansoo
JW
Kim
CureSeq Inc., Brisbane, CA 94005, USA.
Sharon
S
Lee
Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
Jun-Hee
JH
Yoon
CureSeq Inc., Brisbane, CA 94005, USA.
Thomas
T
Kim
CureSeq Inc., Brisbane, CA 94005, USA.
Charles J
CJ
Sailey
Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
David M
DM
Jablons
Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
Il-Jin
IJ
Kim
D005820:Genetic Testing; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016612:Paraffin Embedding; D057089:Pathology, Molecular / Q000379:methods* / Q000592:standards; D016707:Tissue Fixation