RNA-Seq of human HGSC or WES of germline/tumor HGSC: Sample 1468
Germline DNA was extracted from blood or saliva samples according to Gentra Puregene Blood Kit manufacturer instructions. We used sonication fragmentation from Covaris for germline DNA fragmentation and Agilent SureSelectXT Target Enrichment System to prepare libraries from 200-ng gDNA samples per manufacturer's recommended protocols. DNA was extracted from FFPE thin sections derived from archival tissue blocks. Pathologic evaluation of tumor content and location guided the number of slides extracted and the areas of each section containing the highest fraction of tumor cells. Sections selected for extraction were stained with hematoxylin before macrodissection. DNA was isolated using the Qiagen FFPE GeneRead DNA Kit according to manufacturer instructions. DNA quantity was determined using a QubitTM 1X dsDNA HS Assay Kits (ThermoFisher, cat. n. Q33230). We prepared P5 indexed libraries from 20-200ng of Biorupter Ultrasonicator (Diagenode)-sheared FFPE DNA using the KAPA Hyper Library Preparation Kit (Kapa Biosystems). Libraries were assessed for size distribution using the Fragment Analyzer High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies, Inc). Unique P7 indexes and P7 adapters were added to the P5 ligated libraries using 11 cycles of PCR amplification. Herculase II Fusion DNA Polymerase was used. We evaluated libraries for size distribution and quantity using the 4200 TapeStation High Sensitivity D1000 ScreenTape (Agilent Technologies) and the Qubit dsDNA HS Assay Kit (ThermoFisher), respectively. We performed exon target capture using the Agilent SureSelect Clinical Research Exome Enrichment Baits. Following capture, we amplified the exon-enriched libraries using 9 cycles of PCR. We then evaluated the size distribution and quantity using the Agilent TapeStation and Qubit dsDNA HS Assay Kit, respectively. Next, we multiplexed the libraries, 31 samples per pool, and quantified the pools by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems). Sequencing was performed on Illumina HiSeq 4000 with 150 bp paired-end (PE) reads, pooling eight samples per lane.
UTAH-phs003632
Sample_204-150133_HC240-204-150133_S87_L003_001