Minion mk1b sequencer

DNA was extracted from the cerebrospinal fluid of the 11 meningitis patients using Quick DNA Miniprep kit (Zymo Research, CA, USA). The almost complete full‐length 16S rRNA genes were amplified, and barcode sequences were attached using SQK‐RAB201 Rapid Amplicon Barcoding Kit (Oxford Nanopore Technologies). Based on the manufacturer’s protocol, PCR cycling conditions were as follows: Stage 1, 95°C for 1 min; Stage 2, 25 consecutive cycles of 95°C for 20 s, 55°C for 30 s and 65°C for 2 min; and Stage 3, 65°C for 5 min. The amplicon library was sequenced using the MinION Mk1b sequencer and a FLO‐MIN106.1 flow cell (Oxford Nanopore Technologies). Raw sequencing data (fast5 files) were obtained using the offline version of MinKNOW software ver. 1.10.23 (Oxford Nanopore Technologies). Sequencing data at nine different time points (3 min, 5 min, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h and 18 h) from the beginning of sequencing data were obtained for analysis.

PCR products were treated with a ligation kit (SQK-LSK109, Oxford Nanopore Technologies) and a barcoding kit (EXP-NBD114, Oxford Nanopore Technologies). NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End repair/Da-tailing module reagents were prepared according to the manufacturer’s instructions. Barcoded DNA (1 μl) was quantified for the subsequent adapter ligation. DNA library was prepared with 400 ng of barcoded DNA and purified once more prior to sequencing. MinION MK1B sequencer (Oxford Nanopore Technologies) was used for whole genome sequencing with R9.4.1 flow cells. It was important that the flow cell priming should be completed before loading the DNA library. MinKNOW software was used to start the sequencing run for 12 h.

Library preparation was performed using a SQK-LSK108 Sequencing Kit R9.4 version (Oxford Nanopore Technologies, Oxford, UK) using 500 ng of DNA. MinION sequencing was performed using one FLO-MIN106 (R9.4) flow cell with the MinION MK1b sequencer (Oxford Nanopore Technologies). Base-calling and fastq conversion were performed with MinKNOW ver. 1.5.12 followed by poretools or Albacore.

The complete genome of strain Bacillus sp. 1839 was sequenced by the Illumina HiSeq 2500 (Illumina Inc., San Diego, CA, USA) and MinIon (Oxford Nanopore Technologies, Oxford, UK) platforms. Sequencing on the Illumina HiSeq 2500 system was performed at Genoanalytica Company (Moscow, Russia). Genome DNA was fragmented by the Covaris M220 sonicator (Covaris, Woburn, MA, USA). Libraries were constructed using the NEBNext^® Ultra™ II DNA Library Prep Kit for Illumina^® (Illumina Inc., San Diego, CA, USA) for paired-end sequencing, with a target average insert size of 200 bp. For nanopore sequencing, genomic DNA was fragmented by passing through a small gauge needle. The fragmented genomic DNA was used to construct the library with the 1D Genomic DNA by ligation kit SQK-LSK108 following the instructions provided by the manufacturer. Sequencing was carried out on a MinIon Mk1B sequencer (Oxford Nanopore Technologies) using an R9.4.1 Flow Cell. The read qualities were examined by FastQC.

Total RNA was extracted from the leaf samples using a RNeasy Plant Mini Kit (Qiagen, Valencia, CA, United States, product No. 74903). After removal of the relic using RNase-free DNase (Qiagen), the quality of the RNA sample was assessed using 1% agarose gel electrophoresis, a NanoDrop spectrophotometer (ThermoFisher Scientific, Wilmington, DE, United States), and an Agilent 2,100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States). The RNA was then reverse-transcribed to cDNA according to the protocol by Oxford Nanopore Technologies (Jain et al., 2016 (link)). The sequencing libraries were created using a preparation kit (Ultra-Long, Grandomics, Wuhan, China) by fragmenting the cDNA and adding PCR adapters to both ends. After PCR amplification (14 cycles) with LongAmp Taq (NEB, New England Biolabs LTD., Beijing, China), the ONT adaptors were ligated onto the PCR products using T4 DNA ligase (NEB). The ligated PCR products were sequenced on a MinION Mk1B sequencer (Oxford Nanopore, Oxford, United Kingdom). All sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under the data have been released from the SRA database since November 23, 2022.