Validation of a next-generation-sequencing cancer panel for use in the clinical laboratory.
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.
Published In/Presented At
Simen, B. B., Yin, L., Goswami, C. P., Davis, K. O., Bajaj, R., Gong, J. Z., Peiper, S. C., Johnson, E. S., & Wang, Z. X. (2015). Validation of a next-generation-sequencing cancer panel for use in the clinical laboratory. Archives of pathology & laboratory medicine, 139(4), 508–517. https://doi.org/10.5858/arpa.2013-0710-OA
Medicine and Health Sciences
Department of Pathology and Laboratory Medicine