P12_Tumor_PacBio_WGRS
High-molecular-weight (HMW) genomic DNA (gDNA) was extracted using the Promega Wizard HMW DNA Extraction Kit (Promega, Madison, WI, USA), Nanobind panDNA Kit (Pacific Biosciences, CA, USA), or NEB Monarch HMW DNA Extraction Kit (New England Biolabs, Ipswich, MA, USA), depending on the sample type. To minimize mechanical shearing during lysis and purification, wide-bore tips and gentle inversion were utilized throughout the protocol. DNA concentration was determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, MA, USA), and size distribution was assessed on an Agilent 2100 Bioanalyzer with the Genomic DNA ScreenTape (Agilent Technologies, CA, USA) to ensure fragments exceeded 48 kb. For library preparation, gDNA was sheared to an average size of 1520 kb using a Covaris g-TUBE device (Covaris, MA, USA) and purified with SMRTbell Clean-up Beads (Pacific Biosciences, CA, USA). SMRTbell libraries were constructed using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, CA, USA) following the manufacturer's instructions and sequenced on the PacBio Revio system to generate high-fidelity (HiFi) reads (Q20+). 5-Methylcytosine (5mC) at CpG sites was predicted from the HiFi reads using pbjasmine (v.2.2.1), and unaligned BAM files containing methylation data were demultiplexed using lima (v.2.10.0).
SUB16217207
P12_Tumor_PacBio_WGRS