GSM4696913: D1903651 [targeted exome sequencing]; Homo sapiens; OTHER
Genomic DNA extraction  Cell pellets were dissolved in 150µl PBS. Genomic DNA was subsequently extracted by QIAcube (Qiagen) using the QiaAmp Blood mini kit (250, Qiagen) consisting of AL, AE, AW1 and AW2 buffers, Qiagen protease, QIAmp mini spin columns and collection tubes. DNA extraction was conducted according to the manufacturer's instructions. DNA concentrations were measured by using the Dropquant software of the Dropsense. Samples were diluted appropriately and used for exome sequencing, sWGS and ddPCR. Tumor and normal DNA were profiled using the 69-gene MDG SeqCap SOLID panel (https://www.cmgg.be/nl/zorgverlener/labguide/platform-moleculaire-diagnostiek-uz-gent-mdg). This assay involves hybridization of barcoded libraries to custom oligonucleotides (Nimblegen SeqCap) designed to capture all protein-coding exons and select introns of 69 commonly implicated oncogenes, tumor suppressor genes, and members of pathways deemed actionable by targeted therapies. Barcoded sequence libraries were prepared using 100ng genomic DNA with the KAPA HyperPlus Prep Kit (Kapa Biosystems KK8504) and combined in equimolar pools.  The captured pools were subsequently sequenced on an Illumina MiSeq as paired-end 150-base pair reads, producing > 300-fold coverage per ROI.