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 two million non-viable  HG008-T cells (batch 0823p23) using Qiagen's QIAamp Blood Mini Kit (PN: 51104), yielding ~6.4 ug of gDNA as measured by a Qubit Fluorometer and sizing with peak at ~21 kb as measured by an Agilent Fragment Analyzer.  HG008-N-D gDNA was previously isolated from non-viable tissue and quality assessed upon receipt, ~2.5 ug as measured by a Qubit Fluorometer and sizing with peak at ~53 kb as measured by an Agilent Tapestation.PCR-free library preparation was performed using the Illumina TruSeq DNA PCR-Free High Throughput Library Prep Kit (96 samples) (PN: 20015963), starting with one microgram of gDNA sample. Genomic DNA samples were concentration normalized on a Hamilton robot. Intact gDNA was sheared using the Covaris LE220 sonicator. Fragmentation was followed by an initial cleanup and end-repair, which was then followed by a double-sided bead-based size selection of the repaired DNA fragments. Size selection was followed by A-tailing, in turn followed by ligation using unique dual indexed (UDI) adapters (HG008-T:  TTCCTGTT-AAGATACT, HG008-N-D: CGGACAAC-AATCCGGA) to prevent index hopping on patterned flow cells. Two final bead-based clean-ups of the libraries were subsequently performed. The uniquely indexed libraries were then validated using the Fragment Analyzer (Agilent) and pooled based on concentration as determined by Picogreen (ThermoFisher). The final libraries were evaluated for size distribution on the Fragment Analyzer and are quantified by qPCR with adapter specific primers (Kapa Biosystems). The tumor and normal TruSeq libraries were prepared and sequenced approximately four months apart. A single library was prepared for each sample. For each tumor and normal, the TruSeq library was multiplexed and loaded across four lanes of three flow cells to achieve the desired coverage. Paired-end, 2x150bp sequencing, was performed using the Illumina S4 Reagent Kit v1.5 (300 cycles) (PN: 20028312) on an Illumina NovaSeq 6000 (ICS v1.7.5, RTA v3.4.4).
library_name: HG008-N-D_ILMN-PCR-free-3