GSM7653371: gDNA_AB_9; Homo sapiens; OTHER
MagMAXTM Cell-Free DNA Isolation Kit Sequencing libraries were prepared following TruSight Oncology 500 ctDNA Reference Guide (Document # 1000000092559 v00) with the corresponding kit [Illumina Inc. TruSight Oncology 500 ctDNA Kit (48 samples) Cat.# 20039252]. Using End Repair A-Tailing Master Mix, input DNA (30ng for ctDNAs and 40ng for sonicated-gDNAs) was blunt-ended and the 3' ends were A-tailed. Then, UMI1 adapters that contain unique molecular indexes (UMIs) were ligated to DNA fragments. After ligation product clean-up, library fragments were amplified using primers that add index sequences for sample multiplexing. Following this step, a pool of oligos specific to the 523 genes targeted by TruSight Oncology 500 ctDNA panel were hybridized to DNA libraries. In the next step, targeted regions of the enriched DNA libraries were bound with capture probes a second time. The second hybridization ensures high specificity of the captured regions. Then, Streptavidin Magnetic Beads were used to capture the probes hybridized to the targeted regions of interest. Resuspension buffer was used to rinse the captured libraries and remove nonspecific binding from the beads. Finally, enriched libraries were amplified, and a final clean-up step was performed.