Oxford Nanopore Whole Genome Sequencing (std length) of the GIAB HG008-T  tumor cell line (Pancreatic Ductal Adenocarcinoma)
DNA was extracted from approximately five million non-viable  HG008-T cells (batch 0823p23)  utilizing the NEB Monarch HMW DNA Extraction Kit for Tissue (PN: T3060). Total DNA extracted was approximately 80 g. Only 5 g of DNA was sheared using Diagenode's Megaruptor3 (PN: B06010003) with the DNAFluid+ Kit (PN: E07020001) followed by DNA size selection using PacBio SRE kit (PN: 102-208-300). The size of sheared DNA fragments were analysed on Agilent Femto Pulse System using genomic DNA 165 kb kit (PN: FP-1002-0275). Fragment size distribution of post-sheared DNA had peak at approximately 45kb length.The UCSC protocol had been optimized to generate N50s of approximately 30kb with very high throughput for ligation based sequencing. Library preparation was performed with Oxford Nanopore Technologies Ligation Sequencing Kit XL V14 (PN: SQK-LSK114-XL). Enough library was created for 4 library loads onto flow cells.Nanopore based sequencing was performed on Oxford Nanopore Technologies (ONT) PromethION 48/PromethION A Series Data Acquisition Unit (PN: PRO-SEQ048/PRO-PRCAMP) utilizing ONT PromethION R10.4.1 Flow Cells (PN: FLO-PRO114M). Sequencing was performed on the same day as ligation library preparation.  Sequencing was performed utilizing one PromethION R10.4.1 Flow Cell. The flow cell was washed with ONTs Flow Cell Wash Kit XL (PN: EXP-WSH004-XL) each day and re-primed and loaded with fresh library for 4 days of sequencing to maximize throughput.The Dorado v0.3.4 basecaller (https://github.com/nanoporetech/dorado) and model, `dna_r10.4.1_e8.2_400bps_sup@v4.2.0/dna_r10.4.1_e8.2_400bps_sup@v4.2.0_5mCG_5hmCG@v2` 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