Barcoding

The barcoding protocol was executed according to manufacturer’s instructions for native barcoding kit I (EXP-NBD002, ONT) in conjunction with the Nanopore Sequencing kit (SQK-NSK007, ONT) with some modifications (Supplementary file 1A, exp. 4, exp. 5). In brief; 1.5 μg DNA was used as starting material for each sample and vigorously vortexed for 1 min. The DNA sample was end-repaired and dA-tailed using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs [NEB] E7546S; 5 min 20°C, and 5 min 65°C). After an AMPure purification, the DNA fragments were subject to ligation using Blunt/TA Ligase Master Mix (NEB M0367S) for 5 min at 20°C and then 5 min at 65°C. The sample was then purified using AMPure magnetic beads and the DNA was eluted off the beads using 31 μl nuclease-free water. The NB01 and NB02 barcodes were ligated to the fragments of each sample with Blunt/TA Ligase mix (NEB) and incubated for 15 min. After an AMPure purification step, the two samples were pooled. Next, we ligated the adaptor (BAM) and hairpin (BHP) to the barcoded DNA fragments using NEB Quick Ligase (NEB) for 20 min at room temperature (22°C). The HTP (ONT) was added and incubated for another 10 min. The 50 μl MyOne C1 beads were prepared in the incubation step, which tethered the hairpin and ligated DNA fragments. The DNA library was eluted off the beads by ELB (ONT) at 37°C for 10 min and was applied to the flow cell.