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.
Conclusion
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.
Big data from small samples: Informatics of next-generation sequencing in cytopathology
