Element AVITI Short Insert Sequencing of the GIAB HG008-N-P normal pancreatic tissue
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 normal pancreatic tissue (HG008-N-P).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. 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.