title: Illumina 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) was isolated from approximately five million non-viable HG008-T cells (batch 0823p23) and 40 mg of non-viable HG008-N-D 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 quantified using the PicoGreen Assay (Quant-iT PicoGreen , catalog# P11496) and the quality was assessed using 1% agarose gel (Invitrogen E-Gel  Agarose Gels, catalog# G720801).Library preparation was performed for each sample using an in-house PCR-Free protocol, with the Roche Kapa HyperPrep PCR-free Kit (PN: KK8505). Two libraries were prepared for the tumor from  1ug of gDNA for each library and one library was prepared for the normal duodenal sample from 1ug of gDNA.  For both tumor and normal samples, DNA was sheared into fragments of approximately 450-600 bp using a Covaris E220 system. Fragmentation was followed by purification of the fragmented DNA using double-sided bead clean up with different ratios of AMPure XP beads to remove very small and large fragments. DNA end-repair and 3-adenylation were performed in the same reaction followed by ligation of the barcoded adapters using a set of 96 8-bp barcoded adapters from IDT, Illumina TruSeq RNA unique dual (UD) Indexes (PN: 20040870). Post ligation products were cleaned twice with AMPure XP beads to get rid of leftover adapters/adapter dimers.The resulting tumor and normal PCR-free libraries were sequenced on an Illumina NovaSeq 6000 (ICS v1.8.1 and bcl2fastq2 version 2.20.0.422). The tumor libraries were pooled with other samples and loaded in a single lane across four flowcells.  The normal library was sequenced in a pool of samples and loaded in one lane Paired end, 2x150bp sequencing, was performed using the Illumina NovaSeq 6000 S4 Reagent Kit v1.5 - 300 cycles (PN: 20028312) with the  NovaSeq XP 4-Lane Kit v1.5 (PN: 20043131).
library_name: HG008-N-D_ILMN-PCR-free-2