Spoton flow cell r9

Third-generation long-read sequencing was carried out using the MinION device and SpotON flow cell R9.4.1 from Oxford Nanopore Technologies. Sample preparation was carried out using the genomic DNA sequencing kit SQK-LSK109 and the EXP-NBD104 barcoding kit as per the manufacturer’s instructions. Sequencing was carried out using the MinKnow application (version 3.6.0). After 72 h, raw-read Fast5 files were base called using Guppy base caller and reads were demultiplexed using Guppy barcoder (version 3.2.8).
Hybrid genome assemblies consisting of Illumina short reads (forward and reverse reads) and MinION long reads were assembled for each sample using Unicycler (version v0.4.8) [41 (link)]. FastQ files from both platforms were used to perform the assembly. Assemblies were viewed in Bandage (version 0.8.1) to visualize the completed assemblies [50 (link)]. Hybrid genomes were annotated with Prokka [43 (link)]. Genome comparison files were generated using NCBI-blast+ (version 2.12.0) which were visualized using the Artemis Comparison Tool (ACT, version 18.1.0) [51 (link)].

Purification of mRNA from total RNA samples was carried out using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Carlsbad, USA). The subsequent reverse transcription reaction was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Carlsbad, USA). RNA sequencing preparation was carried out using, the Low Input by PCR Barcoding Kit and the cDNA-PCR Sequencing Kit (Oxford Nanopore Technologies, Oxford, United Kingdom), using the MinION Sequencing Device, the SpotON Flow Cell (R9.4.1), and MinKNOW software (Oxford Nanopore Technologies, Oxford, United Kingdom) according to the manufacturer’s instructions. Samples were sequenced for 48 h on two flow cells. Base-calling was performed by Albacore implemented in the nanopore software. Only D²-Reads with a quality score above 8 were used for further alignment.