DNA-Seq of Homo Sapiens: organoid from colon cancer (left) derived from Adult female
Genomic DNA (gDNA) was extracted from fresh frozen colorectal Cancer (CRC) cells using QIAamp DNA Mini Kit (Qiagen), and quantified by Qubit 2.0 Fluorometer (Thermo Fisher Scientific). The mutational status of 22 known genes (KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBX7, FGFR3, NOTCH1, ERBB4, FGFR1, and FGFR2) associated with CRC was screened by Ion PGM platform using the Ion AmpliSeq Colon and Lung Cancer Research Panel v2 (Thermo Fisher Scientific). For each sample, 15 ng of gDNA was amplified according to the protocol for snap frozen samples (PMID: 26633390). Then, 6 barcoded libraries were multiplexed at a dilution of 100 pM and amplified/enriched by OneTouch and the OneTouch ES, respectively (Thermo Fisher Scientific). Each template was loaded into 316v.2 chip and sequenced on Ion PGM (Thermo Fisher Scientific). The sequencing data were analyzed by the Ion Torrent Software Suite by the plugin Torrent Variant Caller v5.10.0.18 (Thermo Fisher Scientific) applying somatic, high stringency parameters. Gene variants were annotated by Ion Reporter Software v.5.16.0.2 and the variant effect prediction was assessed by Cosmic.