Ligation sequencing kit 1d sqk lsk109

Whole genome amplification (WGA) and template preparation was performed following the “Premium whole genome amplification protocol” available in the Oxford Nanopore community. For WGA, all reactions were executed in a BioRad C1000 Thermal Cycler using the REPLI-g Midi Kit (Qiagen), which uses the innovative Multiple Displacement Amplification (MDA) technology. The libraries were performed using the Ligation Sequencing kit 1D SQK-LSK109 and the Flow cell Priming kit EXP-FLP001 (Oxford Nanopore Technologies). All steps performed for library preparation were performed as described by Piñar et al. (2020a) (link). A quality control of flow cells (SpotOn Flow cell Mk I R9 Version, FLO-MIN 106D) was performed prior starting the sequencing. The MinKNOW^TM software was used to check the number of active pores in the flow cells (Table 1) and for performing the sequencing runs. All seven runs were performed for 48 h.

Six NTM isolates, which were obtained from pre- and post- operative culture from 1 refractory patient and 2 recurrent patients, were lysed and extracted DNA using NucleoSpin Microbial DNA (Macherey-Nagel, Duren, Germany) with manufacturer’s instruction. The library was prepared using Ligation Sequencing Kit 1D (SQK-LSK109) (Oxford Nanopore Technologies (ONT), UK) with Native Barcoding Expansion 1–12 (EXP-NBD104) (ONT, UK) according to manufacturer’s instruction. Whole genome sequencing was performed using MinION sequencer with a FLO-MIN106D flowcel (ONT, UK). Raw signal data of fast5 was converted to fastq format by guppy basecaller (v3.5.1) provided by ONT and mapped by minimap2 [28 (link)] using a comparable amount of data in pre- and post- operative condition. Data amount for reccurent case 1 (patient no. 35) and refractory case (patient no. 4) were 200 Mb, for reccurent case 2 (patient no.11) was 22 Mb, respectively. MLST analysis was performed using mlstverse [29 (link)] and the strain name with the highest scores were compared between pre- and post- operatives.