WGS PacBio HiFi for Pan paniscus: adult male B-Lymphocyte cell line
Lymphoblastoid cell lines (LCLs) available at the Coriell Institute for Medical Research were used for Pacific Biosciences (PacBio) HiFi sequencing and expanded to a total culture size of 3x10e6 cells. The cell line expansions were derived from the original expansion culture to reduce the number of passages and minimize culturing time. Cells were washed in PBS and flash-frozen as dry cell pellets of 10e6 cells per vial. High-molecular weight DNA was extracted on 1-3 December 2021 using the Circulomics CBB kit (102-573-600) from a frozen cell pellet (10e6 cells). DNA quantity, purity, and integrity were checked at different steps and at the end of the extraction protocol. DNA quantity was checked on a Qubit Fluorometer I with a dsDNA HS Assay kit (ThermoFisher) and sizes examined on a FEMTO pulse (Agilent Technologies) using a Genomic DNA 165kb kit. Purity ratios were assessed with NanoDrop. A total of 54.8 g of DNA (274 ng/uL in 200 L volume, over 50kb length, and purity ratio 260/280: 1.82, 260/230: 2.0) was used as input for library preparation.  For library prepration, the concentration of the stock genomic DNA (gDNA) was initially measured using the DNA High Sensitivity (DNA-HS) Qubit assay (Invitrogen), and the size of gDNA was estimated using either the Fragment Analyzer or Femto Pulse (Agilent). Approximately 4-5 g of stock gDNA was sheared to a target size of 20-30 kb using a Megaruptor 3 instrument (Diagenode). Subsequently, the sheared DNA underwent size selection using a Pippin HT instrument (Sage Science) to target a size range of 15-22 kb. Following size selection, the DNA was used for CCS (Circular Consensus Sequencing) library preparation using the SMRTBell Express Template Prep Kit 2.0 and Enzyme Cleanup Kit 1.0 (PacBio). Each library was barcoded using PacBio Barcoded Overhang Adapters. Post-library preparation, the concentration of the DNA stock was measured using the DNA-HS Qubit assay, and the DNA size was estimated using the Fragment Analyzer or Femto Pulse. Sequencing was conducted on a Pacific Biosciences Sequel IIe instrument, using version 2.0 sequencing reagents and operating on instrument control software version 10.1.0.119549, with a movie collection time of 30 hours for each of 10 8M SMRTCells with no pre-extension and with adaptive loading. CCS/HiFi reads were generated from the initial subread data using the CCS program version 6.0.0 within PacBio SMRTLink version 10.1.0.119588. Fastq sequences were extracted from BAM files using SMRTLink 10.1.0.119588.