Next-Generation Sequencing (NGS) is a powerful tool to analyze genome changes. With falling cost, NGS is more suitable to be a diagnosis tool in clinical applications. However, the performance of diagnostic tests must be verified and validated in a clinical diagnostic laboratory. We evaluated the applicability of the Ion AmpliSeq Cancer Hotspot Panel v2 using Ion Proton Sequencer for screening 50 cancer-related genes in fresh tumor tissues.
Methods
DNA was extracted from the fresh tissue biopsy of colorectal tumor. Use the Ion AmpliSeq Cancer Hotspot Panel v2 and Ion Proton Sequencer for screening hotspot regions of 50 oncogenes and tumor suppressor genes. The same sample was detected repeatedly by this panel for six times. We compared the concordances and differences data between six tests, and all mutation variants were confirmed by Sanger
sequencing. We evaluated the clinical validation of this panel.
Results
The average Ion Proton sequencing output per run was 28.9 megabases with 0.25 million sequencing reads. The reproducibility of this panel counted from six tests was 94.42%. In total, the Ion Proton detected 20 expected mutation variants, and these variants were related to 14 genes. In these 20 variants, 13 variants were detected in every test, 2 variant were detected in three times and twice respectively, and 5 variants only appeared once. All of the variants appeared less than four times were confirmed to be false, but 5 of 13 variants which were detected in every test were also confirmed to be false by Sanger sequencing. Two false variants* were caused by the defect of method due to homopolymers (>6 identical bases). The true mutation variants were all from novel mutations (not include in designed hotspots), but three mutation variants from hotspots were all false. In addition, the high coverage variant# was also proved to be false. From these findings, it’s hard to distinguish the variant is true or false only by few quality data. We only calculated the mutation variants detected in six times, but the false variant rate was still 38.5%.
Conclusions
This panel can simultaneously screen 50 cancer-related genes covering 2,855 COSMIC mutations with a low input of DNA .It's a convenient and powerful tool with reduced time and cost of genetic analysis to implement in clinical diagnosis. We revealed the risk of false variants with high reproducibility and high coverage. Therefore, we suggest that it's necessary to verify any variant for the clinical use of the Ion Ampliseq cancer hotspot panel v2.
