Nextera xt v2

Sequencing libraries were generated from the 16 middle fractions of each sample using Nextera XT v2 chemistry (Illumina) with 12 PCR cycles. Concentrations and size distributions of each library were determined on a Fragment Analyzer (Agilent). Libraries were pooled at equal molar concentrations within the range of 400–800 bp, and the pool was size selected to 400–800 bp using a Pippin Prep 1.5% agarose, dye-free, internal marker gel cassette (Sage Science). For each library, 2 × 150 bp paired-end sequencing was performed on the Illumina Novaseq platform using S4 flowcells (Table S6 at https://doi.org/10.6084/m9.figshare.22280632).

Whole blood was collected to K2E (EDTA) vacutainers (Becton Dickinson) and donors’ genomic DNA was extracted from 1 ml of whole blood by the salting‐out method. The DNA sample purity and concentrations were measured by NanoDrop ND‐1000 spectrophotometry. Genomic DNA (500 ng) was treated with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research Corporation) according to the manufacturer's instructions. Bisulfite treated DNA was amplified in 10 ul reaction containing 0.72 ng of DNA, 1X Yellow PCR Buffer with (NH4)2SO4 (Naxo), 1.5mM MgCl₂ (Solis BioDyne), 2 mM dNTP mix (Solis BioDyne), 0.2 µM of each primer, and 0.06 U HOT FIREPol DNA Polymerase (Solis BioDyne). Cycle conditions were as follows: 95°C for 15 min, 1 cycle; 40 cycles (95°C for 20 s, 56°C for 30 s, 72°C for 1 min); and 72°C for 3 min, 1 cycle. Primer sequences used in PCR reactions can be delivered on request by the authors. Amplicons from each individual were combined in equal amounts, purified with Agencourt AMPure XP beads (Beckman Coulter) and labeled with Nextera XT v2 (Illumina) indexes. Paired‐end sequencing of bisulfite‐treated DNA with read length of 250 bp was done with Illumina MiSeq at the Core Facility of the Institute of Genomics of the University of Tartu.

For Illumina MiSeq, genomic libraries were prepared using Nextera XT V2 (Illumina, United States), with bead-based normalization. Paired-end WGS was performed using the V3 kit. Each sequencing run was multiplexed up to 48 isolates to provide > 20X coverage.
For ONT, gDNA (Qiagen) was concentrated and purified at a 1:1 ratio using SPRI beads (Mag-Bind TotalPure; Omega) with 15 μl water elution. Genomic libraries were prepared using the Rapid Barcoding Kit (SQK-RBK004; ONT) and sequenced on a FLO-MIN106 R9.4 flow cell using a MinION (ONT). Sequencing was performed on single-use MinION flow cells for a running time of 72 h with default parameters within MinKNOW unless otherwise specified.
We compared yield and LRS metrics between R9 and R10 flow cells. gDNA (n = 53) was extracted using the Qiacube, concentrated using SPRI beads, and libraries generated using the 96-Rapid Barcoding Kit (SQK-RBK110.96; ONT). gDNA was pooled and divided into 6 aliquots for simultaneous flow cell loading onto three flow cells; two R10 (one on a MinION Mk1B connected to an Intel i7-8750H laptop, another on a MinION Mk1C) and one R9 (Mk1B connected to an Intel i7-6700 desktop computer). Following the initial sequencing period, flow cells were washed (WSH003), QC checked to determine recovery of nanopores, and re-loaded with the remaining aliquots of gDNA (stored at 4°C).