High-Molecular-Weight DNA Extraction and HiFi Genome Assembly

High molecular weight DNA was extracted from blood cells using a NucleoBond AXG column (Macherey-Nagel, Düren, Germany), which was followed by purification with phenol–chloroform. The concentration of the extracted DNA was measured with Qubit 4 (ThermoFisher, MA, USA), and their size distribution was first analysed with TapeStation 2100 (Agilent Technologies, CA, USA) to ensure high integrity and later analysed with pulse-field gel electrophoresis on CHEF DR-II (BioRad, CA, USA) to ensure the size range between 20 kb and 100 kb. The DNA was fragmented with g-TUBE (Covaris, MA, USA) and size-selected with BluePippin according to the official protocol. A SMRT sequence library was constructed with an SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA) and was sequenced in a single 8M SMRT cell on a PacBio Sequel IIe system (Pacific Biosciences). The sequencing output was processed to generate circular consensus sequences (CCS) to obtain a total of 33.7 Gb HiFi sequence reads (Accession ID, DRR486909). From these reads, adapter sequences were removed using the program HiFiAdapterFilt.^{8 (link)} The obtained HiFi sequence reads were assembled using the program hifiasm v0.16.1^{9 (link)} with its default parameters. The obtained contigs were subjected to haplotig purging by using the program purge_haplotigs¹⁰ with the options ‘-l 5 -m 23 -h 45’.