title: PacBio Onso Whole Genome 2x150  Sequencing of the GIAB HG008-N-D normal duodenal tissue
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 normal duodenal tissue (HG008-N-D). 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.PCR-free library preparation was performed using the PacBio Onso fragmentation DNA library prep kit (PN: 102-499-100). Two libraries were prepared, one for each of the tumor (HG008-T) and normal duodenal (HG008-N-D) samples starting with 200 ng of high molecular weight (HMW) gDNA.   HG008-T and HG008-N-D HMW genomic DNA (gDNA) were first enzymatically fragmented followed by end-repair, A-tailing and adapter ligation per the fragmentation kit protocol.  A small aliquot of libraries were amplified using the PacBio Onso library amp kit (PN: 102-410-800) for library QC purposes (library size measurement by Agilent TapeStation). Quantitative PCR was performed with the PacBio Onso library quant kit (PN: 102-431-800) to quantify Onso-compatible libraries for flow cell loading. Whole genome sequencing of the resulting PCR-free libraries was performed on a PacBio Onso system using sequencing-by-binding (SBB) technology. The tumor library was loaded across four lanes of two flow cells and the normal duodenal library across two lanes of one flow cell. Cluster generation and paired-end 2x150 bp reads were generated using the PacBio Onso 300 cycle sequencing kit (PN: 102-860-300).  Onso reverse (R2) reads may contain 1bp reads.  It is advised these reads be filtered out prior to further processing using a suitable trimmer such as cutadapt (http://dx.doi.org/10.14806/ej.17.1.200)
library_name: HG008-N-D_PB-Onso-1