Nanopore Sequencing of Microbial Genes

The sequencing libraries were prepared using the Native barcoding amplicons protocol with EXP-NBD104 and SQK-LSK109 kits (Oxford Nanopore Technologies) according to Davidov et al. (2020) (link). The 16S rRNA and 18S rRNA gene sequencing libraries were loaded to the same MinION flow cell in two batches: the first included the amplified 16S rRNA and 18S rRNA gene DNA barcodes of replicates 1, 3, and 5 (12 multiplexed libraries in total), and the second included the amplified 16S rRNA and 18S rRNA gene DNA barcodes of replicates 2 and 4 (8 multiplexed libraries in total). The two sequencing runs were separated by a washing step using EXP-WSH002 Kit (Oxford Nanopore Technologies). Each library was loaded onto to the MinION Nanopore Spot-on flow cell (FLO-MIN106D, version R9) and sequenced until reaching ∼7 Giga nucleotides (∼4 M reads). The ITS2 sequencing library was loaded to a new MinION flow cell and sequenced until reaching ∼1.3 Giga nucleotides (∼1 M reads). Base-calling for all libraries were done by the Guppy base calling software 3.3.3, using MinKNOW program with the “high accuracy” option. Raw reads were obtained in FAST5 and FASTq formats from which “pass” quality reads were subjected to further analysis.