Oxford Nanopore Ultra Long Whole Genome Sequencing of the GIAB HG008-T  tumor cell line (Pancreatic Ductal Adenocarcinoma)
Ultra-high molecular weight DNA was extracted from approximately five million non-viable HG008-T cells (batch 0823p23) utilizing New England Biolabs Monarch HMW DNA Extraction Kit for Tissues (NEB #T3060). Wide bore pipette tips were used throughout to preserve ultralong DNA. No QC of resulting gDNA was performed as viscosity of isolated DNA solution is challenging to work with. Rather ultra long DNA is assessed as part of the ONT sequencing and previous experience with a five million cell as input.Library preparation was performed utilizing Oxford Nanopore Technologies Ultra-Long DNA Sequencing Kit Version 14 (SQK-ULK114). The library was eluted in approximately 810 L volume in order to sequence across additional flow cells to maximize data collection.Nanopore based sequencing was performed on Oxford Nanopore Technologies (ONT) PromethION 48/PromethION A Series Data Acquisition Unit (PN: PRO-SEQ048/PRO-PRCAMP) utilizing ONTs PromethION R10.4.1 Flow Cells (PN: FLO-PRO114M). Sequencing was performed the day following library prep. Sequencing was performed utilizing three PromethION R10.4.1 Flow Cells. The flow cells were washed with ONTs Flow Cell Wash Kit XL (EXP-WSH004-XL) each day and reloaded with library for 4 days of sequencing to maximize throughput. The Dorado v0.4.3 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.