Nebnext companion module for technologies ligation sequencing

Wolbachia DNA was extracted from a whole Drosophila female (150 flies for one sample) or its ovaries (150 pairs for one sample), following the protocol described in [55 (link)].
For MiSeq sequencing, 1 mkg DNA from whole flies was fragmented using a Covaris M220 sonicator with parameters optimized for a maximum fragment-size of approximately 400 bp. Barcoded-genome libraries were prepared, using 50 ng of fragmented DNA, with Roche KAPA Hyper Prep Kit, KAPA UDI adapters, according to the manufacturer’s protocol for dual size-selection. The amplification of libraries was carried out in 9 cycles. The quality and molarity of the libraries were determined using a Bioanalyzer BA2100 and Qubit fluorim-eter. After normalization, the barcoded libraries were pooled and sequenced, using the MiSeq Reagent Kit v2 (500-cycles).
For Nanopore sequencing, 1 mkg DNA was fragmented by pipetting five times in 20 mkl volume. Libraries were prepared using the NEBNext^® Companion Module for Oxford Nanopore Technologies^® Ligation Sequencing (Oxford Nanopore Technologies, Oxford, UK), according to the manufacturer’s protocol without barcoding, and using a long-fragment buffer at the washing step. Nanopore sequencing was performed using the MinION Mk1C device, SpotON Flow Cell (R9.4) and Ligation Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK).

The amplicons were purified with CleanNGS magnetic beads (Labgene Scientific, Switzerland) and quantified with the Qubit 4 system (Thermo Fisher Scientific, USA). Twenty-four ng (2 pmol) of purified amplicon DNA were used for the barcoding procedure, using barcodes from the PCR Barcoding Expansion 1–96 kit (ONT, UK), and the amplification was carried out with the 2 × KAPA HiFi Hot Start Ready Mix kit (Roche, Switzerland). The cycling conditions were: first denaturation of 3 min at 95 °C, followed by 13 cycles of denaturation of 20 s at 95 °C, annealing of 15 s at 62 °C, elongation of 30 s at 72 °C, and a last elongation of 5 min at 72 °C. The barcoded DNA samples were subjected to a second step of magnetic beads purification and quantification, using the same methods as above, and combined in equal ratios to generate a pool.
One µg of pooled barcoded libraries was finalised using the Ligation Sequencing kit (ONT, UK) and the NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing (New England Biolabs, USA). After a last step of magnetic beads purification and quantification, the libraries were loaded onto a R9.4 flow cell (ONT, UK) using the Flow Cell Priming Kit (ONT, UK).