title: Element AVITI Short Insert Sequencing of the GIAB HG008-T  tumor cell line (Pancreatic Ductal Adenocarcinoma)
design: Sequencing was performed on the HG008-T pancreatic ductal adenocarcinoma tumor cell line with matched normal duodenal tissue (HG008-N-D) and 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 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.Library preparation was performed using isolated genomic DNA (gDNA)  from non-viable HG008-T cells (batch 0823p23) and non-viable HG008-N-D and HG008-N-P tissue.  One short insert library (350-400 bp) was prepared from each gDNA source.  For each library, 500 ng  of gDNA was input into the Element Elevate Mechanical Library Prep Kit (PN: 830-00008, Document # MA-00004 Rev. E) using the Elevate Long UDI Adapter Kit Set A (PN: 830-00010).  Mechanical shearing of gDNA was performed using a Covaris ultra-sonicator to achieve the desired insert size of ~400 bp. For the short insert libraries seven iterations with the following settings were performed: 10 second duration, peak power 50, duty cycle 20, 1000 cycles per burst, average power 10. Shearing was followed by end repair and A-tailing.  PCR-free library preparation continued with adapter ligation, generating dual-index libraries. Size selection was performed with double-sided SPRI bead clean-up with SPRI ratios of 0.46X/0.62X. Linear short insert libraries were quantified for loading onto the AVITI system using qPCR.Linear libraries were loaded onto individual high output flowcells, 6 flowcells in total (3 for short insert, 3 for long insert). The Element AVITI 2x150 Sequencing Kit Cloudbreak UltraQ sequencing chemistry (PN: 860-00018) was used to generate 150bp paired-end reads on the AVITI system.