title: PacBio Revio Whole Genome Sequencing of the GIAB HG008-T  tumor cell line (Pancreatic Ductal Adenocarcinoma)
design: Sequencing was performed on the HG008-T pancreatic ductal adenocarcinoma tumor cell line with matched normal duodenal tissue (HG008-N-D), using similar methods and library preparation protocols tailored for both tumor and normal samples as described below. The data presented here corresponds to the tumor cell line (HG008-T). Genomic DNA (gDNA) was isolated from approximately five million non-viable HG008-T cells (batch 0823p23) and 40 mg of non-viable HG008-N-P tissue using the Revvity chemagic Prime 8 Instrument.  Cells were resuspended and homogenized in the tissue lysis buffer, vortexed, and treated with RNase. The homogenate was then transferred to the chemagic Prime 8 instrument where DNA was extracted using the chemagic Prime DNA Blood 2k Kit H24 kit (PN: CMG-1497). For the normal tissue sample, the tissue was minced with a razor blade, incubated overnight in the lysis buffer with proteinase K. The following day the homogenate was subjected to an RNase treatment and transferred to the chemagic Prime 8 instrument where DNA was extracted using the chemagic Prime DNA blood extraction kit (CMG-1497). Genomic DNA mass yields for tumor and normal samples were approximately 68 and 72 ug, respectively. Resulting DNA size was measured on an Agilent Femto Pulse System with average sizing of approximately 81kb for gDNA from tumor cell line and 59kb for gDNA from normal duodenal tissue.Genomic DNA (7.5ug) was sheared using Covaris g-tubes to achieve an average size of 15-20 kb.  Sheared DNA was size selected with a Sage Science PippinHT using the 15-20 kb high-pass 75E cassette definition (gel cassette HPE7510) to eliminate fragments smaller than 15 kb.  One library for each sample type was prepared. The PacBio procedure for library preparation was performed using the PacBio SMRTbell prep kit 3.0 (PN: 102-182-700) and the SMRTbell adapter index plate 96A (PN 102-009-200) for  repair and A-tailing, adapter ligation and nuclease treatment to remove unligated fragments.  Final libraries were purified using 1X PacBio SMRTBell Cleanup beads, eluted in 30 L PacBio elution buffer and quantified using the Qubit dsDNA quantification high-sensitivity assay (Thermo Fisher Scientific). Final library size was determined using the Agilent Femtopulse.PacBio SMRTbell libraries were prepared for HiFi sequencing (i.e. circular consensus sequencing) by annealing and binding sequencing primers and polymerase, respectively, to the SMRTbell templates using Revio polymerase kit (PN: 102-817-600) and following instructions provided in the Sample Setup module of PacBio SMRT Link software (13.0.0.207600).  Sequencing-ready SMRTbell libraries were sequenced on a PacBio Revio system (ICS version 13.0.0.212033). 30 hour movies were run. Two SMRT cells were run for HG008-T and one SMRT cell for HG008-N-D. QC metrics from SMRTlink software including HiFi reads, mean read length, P1%, etc were as expected.
library_name: HG008-T_PB-HiFi-2