Pacbio sequel iie system

An overnight culture of S. suis was diluted 500× in fresh pre-warmed THY and incubated at 37°C until it reached an OD of 0.30–0.45. Genomic DNA was isolated using the Wizard genomic DNA purification kit (Promega) according to manufacturer’s protocol. DNA was quantified with Qubit, and DNA integrity was assessed on a 0.7% agarose gel. For detailed sequencing methods, see supplemental materials. Briefly, sequencing libraries were constructed from sheared genomic DNA (5–20 kb) and sequenced on a SMRT Cell 8M using a PacBio sequel II(e) system (Pacific Biosciences).

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’.