Flow cell wash kit

MiSeq libraries were prepared with 1 ng of genomic DNA input using the Nextera XT DNA Library Preparation Kit (Illumina Inc., SanDiego, CA) following the manufacter’s instructions. Afterwards, libraries were sequenced using the MiSeq Reagent Kit v2 (500-cycles) (Illumina Inc.) on a MiSeq System using the 2x250 bp pair-end chemistry. The adapter trimming option in the Illumina FASTQ file generation pipeline was used to remove adapter sequences from the 3’ ends of the reads.
For MinION sequencing, libraries were prepared using the Ligation Sequencing Kit (Oxford Nanopore Technologies Inc., Oxford, UK) using the 1D Genomic DNA by Ligation protocol (SQK-LSK109), with a minor modification that 4 μg of genomic DNA input was added as the initial input instead of the recommended amount of 1 μg. The prepared libraries were loaded into a MinION flow cell (R9.4.1) and sequenced on the MinION device. The sequenced reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. After each use, flow cells were washed using the Flow Cell Wash Kit (Oxford Nanopore Technologies Inc.) and stored at 4°C. Flow cells were reused if the available pore number were above 800 based on the quality check.

For the prepared ONT library sequencing, MinION Flow Cell (R9.4.1, FLO-MIN-106D, Oxford Nanopore Technologies, Oxford, UK) with MinKNOW software (v21.06.13, Oxford Nanopore Technologies) was utilized. FAST5 raw data were collected for up to 48 h for each run, simultaneously processing the FAST5 data to the FASTQ file using a GPU-based ont-guppy basecaller (v5.0.16, Oxford Nanopore Technologies) in super-accurate mode with barcode and adaptor trimming, until the number of passed reads for the barcode of the lowest value exceeded 100,000. As the sequencing capacity of MinION Flow Cells was much higher than the maximum of 24 samples that could be multiplexed with the SQK-16S024 barcoding kit, after reaching the desired throughput for a sequencing run, the MinION cells were washed with the Flow Cell Wash Kit (EXP-WSH004, Oxford Nanopore Technologies) after each run, stored, and reused until the pores were depleted. For the Illumina V3–V4 library, the MiSeq platform was used to sequence the index-attached V3–V4 amplicons under 300 × 2 paired-end read conditions using standard Illumina 16S Metagenomic Sequencing Library protocols to obtain FASTQ data of read count ranging from 121,470 to 609,040.