Oxford Nanopore Whole Genome Sequencing (Std length) of the GIAB HG008-N-P normal pancreatic tissue
Sequencing was performed on normal pancreatic tissue (HG008-N-P) and normal duodenal tissue (HG008-N-D), using similar methods and library preparation protocols tailored for both 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-N-P and HG008-N-D tissues with Oxford Nanopore Technologies Ligation Sequencing Kit V14 (PN: SQK-LSK114) using one microgram of each gDNA.Nanopore based sequencing was performed on Oxford Nanopore Technologies (ONT) PromethION utilizing ONTs PromethION R10.4.1 Flow Cells (PN: FLO-PRO114M). To maximize throughput multiple flow cells were used. Sequencing was performed using four flow cells for the duodenal sample and three flow cells for the pancreatic sample. The Dorado v0.5.3 basecaller (https://github.com/nanoporetech/dorado) and DNA model, `dna_r10.4.1_e8.2_400bps_sup@v4.3.0_5mC_5hmC@v1`were used for basecalling, including modified bases. Basecalling successfully completed with no errors. Output from the basecaller is a bam file of unaligned reads including modified bases.