Validating Cancer Variant Detection Using Hotspot Control

The Acrometrix Oncology Hotspot Control DNA (Thermo Fisher, Waltham, MA; Cat. No. 969056) was used for generating sequencing data for testing and evaluating MGE and ELECTRO as previously described [22 (link)]. This control DNA contains more than 500 COSMIC mutations from 53 genes (Thermo Fisher, Waltham, MA; Cat. No. 969056). A targeted cancer panel with selected 25 genes (NextDay Seq-Pan Cancer HotSpot Panel kit, CureSeq Inc.) was used for a deep sequencing of the The Acrometrix Oncology Hotspot Control DNA as previously described [22 (link),34 (link)]. In brief, 10 ng of control DNA was amplified and ligated with barcoded and adaptors with the library prep kit (NextDay Seq-Pan Cancer HotSpot Panel kit, CureSeq Inc.). The prepared library was cleaned up using magnetic beads and resuspended in 1x low-TE buffer, followed by qualitative and quantitative electrophoretic analysis by High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA; Cat. No. 5067–4626). After the emulsion PCR and library enrichment, the prepared library was sequenced using an Ion 314 v2 Chip (Thermo Fisher), and sequenced on an Ion Torrent™ PGM sequencer. The generated sequencing data was processed with MGE from data submission to data distribution as described above.