PacBio Onso Whole Genome 2x150 Sequencing of the GIAB HG008-T tumor cell line (Pancreatic Ductal Adenocarcinoma)
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 the non-viable 0823p23 batch of cells of the GIAB HG008-T tumor cell line using the Qiagen Puregene Cell Kit (PN: 158767). Approximately five million cells were input into the Qiagen Puregene protocol. Briefly, cells were lysed, treated with RNase, gDNA precipitated, washed and hydrated in TE buffer (1 mM EDTA, 10 mM TrisCl pH 7.5). The gDNA was viscous and was therefore allowed to homogenize for one week at ambient laboratory temperature.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)