MinION library preparation and sequencing

MinION Library preparation was performed according to the native barcoding genomic DNA protocol (EXP-NBD104, EXPNBD114, and SQK-LSK109)²⁶ with minor modifications. The library was prepared using the Ligation Sequencing Kit (ONT; SQK-LSK109). For each run, first, DNA for each sample was repaired and end-prepped for each sample using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, Inc.; Catalog # E7546S). 90 μL AMPure XP beads were used for cleaning up repaired DNA. Then repaired DNA was washed on a magnetic rack using freshly made 70% ethanol and eluted with 25 μL nuclease-free water. Second, native barcode ligation was performed by mixing 22.5 μL of the elute with the Blunt/TA Ligase Master Mix (New England Biolabs, Inc.; Catalog # M0367S) and Native Barcode (ONT; Native Barcoding Expansion Kit EXP-NBD104). Barcoded DNA was cleaned up by another wash step using 90 μL AMPure XP beads, and DNA was eluted in 26 μL nuclease-free water. Then equimolar amounts of each barcoded DNA were pooled into a 1.5 mL microcentrifuge tube. Last, adapter ligation was performed by mixing the pooled barcoded sample with Adapter Mix (ONT; SQK-LSK109), NEBNext Quick Ligation Reaction Buffer (New England Biolabs, Inc.; Catalog # B6058S) and Quick T4 DNA Ligase (New England Biolabs, Inc.; Catalog # M2200S). Ligated DNA was cleaned up with 60 μL AMPure XP beads, washed on a magnetic rack using Long Fragment Buffer (ONT; SQK-LSK109), and eluted with 15 μL Elution Buffer (ONT; SQK-LSK109).

Sequencing reactions were performed independently for each run on a MinION flow cell (ONT; FLO-MIN106 R9.4.1 Version) connected to a Mk1B device (ONT; MIN-101B) operated by the MinKNOW software (ONT, Inc. v19.12.2). Each flow cell was primed with the priming buffer prepared by mixing 30 μL Flush Tether (ONT; EXP-FLP002) with a tube of Flush Buffer (ONT; EXP-FLP002). 12 μL of the final library mixed with Sequencing Buffer (ONT; SQK-LSK109) and Library Loading Beads (ONT; SQK-LSK109) were loaded onto the SpotON sample port of the flow cell in a dropwise fashion. The sequencing run was stopped when all pores lost activity, usually after 48–72 h. A new flow cell was used for each run. Sample IDs and descriptions are shown in Table ​Table1.1. After sequencing, the raw files in FAST5 format, containing the electrical signals, were translated (base-called) with the ONT tool Guppy GPU (v3.2.2) into sequences with a minimum q-score of 7 and saved as FASTQ files for further analysis. The FASTQ files were then converted to FASTA files with an in-house shell script.