title: PacBio Revio Whole Genome Sequencing of the GIAB HG008-N-P normal pancreatic tissue
design: Sequencing was performed on the HG008-T pancreatic ductal adenocarcinoma tumor cell line with matched normal pancreatic tissue (HG008-N-P), using similar methods and library preparation protocols tailored for both tumor and normal samples as described below. The data presented here corresponds to the normal pancreatic tissue (HG008-N-P).Genomic DNA (gDNA) from non-viable normal tissue was isolated using the Qiagen Puregene Tissue Kit (PN: 158667).  Approximately 2 ml of Qiagen kit cell lysis buffer with 30 ul of Proteinase K was added to 10-15 mg of tissue. A Dounce Homogenizer was used to disrupt the tissue before leaving it overnight at 55C. This was followed by addition of RNase A and another incubation at 37C for 5 minutes followed by cooling on ice. Genomic DNA was precipitated, washed and hydrated in TE buffer (1 mM EDTA, 10 mM TrisCl pH 7.5) per the Qiagen Puregene protocol. Library preparation was performed using isolated genomic DNA (gDNA) from non-viable HG008-T cells (batch 0823p23) and non-viable HG008-N-D tissue. Sizing of the isolated gDNA was performed prior to library preparation to confirm the gDNA was of sufficient length for long-read sequencing. The average sizing of smear analysis from Pulsed Field Gel Electrophoresis (PFGE) with an Femto Pulse System (Agilent) was approximately 58kbp for HG008-T and 29kbp for HG008-N-P.Library preparation was performed following PacBio procedure and checklist:  "Preparing whole genome and metagenome libraries using SMRTbell prep kit 3.0" (PN: 102-166-600 REV02 MAR2023). This protocol uses the PacBio SMRTbell prep kit 3.0 (PN: 102-182-700), SMRTbell adapter index plate 96A (PN: 102-009-200), and SMRTbell cleanup bead kit (PN 103-306-300). Briefly, 6.4 ug of HG008-T and 6.1 ug of HG008-N-P were sheared separately on a Megaruptor 3 (Diagenode, B06010003) to a target size of 15-20kb. Two library preparations were performed using sheared gDNA inputs of 3.1 ug of HG008-T and 4.1 ug of HG008-N-P.  Sheared samples were subjected to a combined DNA repair and A-tailing reaction followed by ligation of sequencing adapters to create SMRTbell libraries. SMRTbell libraries were treated with a nuclease mix to remove unligated library components and then size-selected on a Sage Science PippinHT system using a 0.75% agarose gel cassette with cassette definition file, "610kb High Pass Marker 75E to remove fragments <10 kb.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 (v13.0), a web-based end-to-end sequencing workflow manager. Sequencing-ready SMRTbell libraries were sequenced on a Revio system using the following reagents and consumables:  Revio sequencing plate (PN 102-587-400) and Revio SMRT Cell tray (PN 102-202-200). 24 hour movies were run. Two SMRT Cells were run for HG008-T and one SMRT Cell for HG008-N-P.
library_name: HG008-N-P_PB-HiFi-1