Guppy v 3

Raw nanopore data were base-called using Guppy v3.2.2 (Oxford Nanopore Technologies, Oxford, UK). After quality filtering using NanoFilt (De Coster et al., 2018 (link)) and residual adapter removal using Porechop,² the obtained dataset was quality checked using NanoPlot (De Coster et al., 2018 (link)). Long nanopore reads were then assembled in hybrid mode using Unicycler (Wick et al., 2017 (link)). The remaining ambiguities in the genome assemblies were verified by PCR amplification of DNA fragments, followed by Sanger sequencing with an ABI3730xl Genetic Analyzer (Life Technologies) using BigDye Terminator Mix v. 3.1 chemistry (Life Technologies). All of the possible sequence errors and mis-assemblies were manually corrected using Seqman software (DNAStar) to obtain the complete nucleotide sequence of bacterial genomes.

Raw nanopore data was basecalled using Guppy v3.2.2 (Oxford Nanopore Technologies, Oxford, UK). After quality filtering using NanoFilt [50 (link)] and residual adapter removal using Porechop (https://github.com/rrwick/Porechop), the obtained dataset was quality checked using NanoPlot [50 (link)]. Long nanopore reads were then assembled using Flye v2.6 [51 (link)]. Flye assembled contigs were further polished using Illumina sequencing reads and Unicycler_polish pipeline (https://github.com/rrwick/Unicycler).

Libraries for nanopore sequencing were prepared using the SQK-LSK109 ligation kit and loaded into a FLO-MIN106 R9 flow cell. Fast5 files were base called, demultiplexed, and adapter trimmed using Guppy v3.2.1 (Oxford Nanopore Technologies, Oxford, UK) with the high-accuracy model and a minimum quality score of 7, obtaining a final approximate coverage of 245× per strain.
The genomes of O. oeni strains AWRIB429, AWRIB787, and ATCC BAA-1163 were assembled and circularized using Unicycler v.0.4.8 (70 (link)) and then polished with long reads using Racon v.1.4.13. A final polish was performed in the genome sequence of strain AWRIB429 with Pilon v.1.23 (71 (link)) using 2 × 150-bp synthetic Illumina reads obtained from the genome assembly of this strain available in NCBI (assembly accession GCA_00017535). Gene and functional annotations were performed with PROKKA v.1.14.16 (72 (link)), including the gene models and functional annotations of O. oeni PSU-1 (assembly accession GCF_000014385). Further functional protein annotations were performed with KEGG (73 (link)) and InterProScan 5 (74 (link)). The prediction of temperate bacteriophages was performed using PHASTER (75 (link)).

Libraries for nanopore sequencing were prepared using the SQK-LSK109 ligation kit and loaded into a FLO-MIN106 R9 flow cell. Fast5 files were base called, demultiplexed, and adapter trimmed using Guppy v3.2.1 (Oxford Nanopore Technologies, Oxford, UK) with the high-accuracy model and a minimum quality score of 7, obtaining a final approximate coverage of 245× per strain.
The genomes of O. oeni strains AWRIB429, AWRIB787, and ATCC BAA-1163 were assembled and circularized using Unicycler v.0.4.8 (70 (link)) and then polished with long reads using Racon v.1.4.13. A final polish was performed in the genome sequence of strain AWRIB429 with Pilon v.1.23 (71 (link)) using 2 × 150-bp synthetic Illumina reads obtained from the genome assembly of this strain available in NCBI (assembly accession GCA_00017535). Gene and functional annotations were performed with PROKKA v.1.14.16 (72 (link)), including the gene models and functional annotations of O. oeni PSU-1 (assembly accession GCF_000014385). Further functional protein annotations were performed with KEGG (73 (link)) and InterProScan 5 (74 (link)). The prediction of temperate bacteriophages was performed using PHASTER (75 (link)).

DNA samples of affected individuals with OPDM and healthy individuals were sequenced using PromethION sequencer (Oxford Nanopore Technologies). Library preparation was carried out using a 1D Genomic DNA ligation kit (SQKLSK109) according to the manufacturer’s protocol. For each individual, one PRO-002 (R9.4.1) flowcell was used. Data analysis was followed by the pipeline in our previous work [18 (link)]. Briefly, PromethION data base-calling was performed using guppy v.3.3.0 (Oxford Nanopore Technologies), and only pass reads (qscore ≥ 7) were used for subsequent analysis. Long reads were aligned to reference genome (GRCh37/hg19) by minimap2 [19 (link)]. To validate the accuracy of ONT, we also performed PacBio Single Molecule, Real-Time (SMRT) DNA sequencing for patients FIII-39. HQRF (High quality Region Finder) were used to identify the longest region of singly-loaded enzyme activity. low-quality areas were filtered by Signal Noise Ratio (SNR). Subreads were obtained after the basic filtrations as previous reported. Circular Consensus Sequence (CCS) reads were retained using CCS tools and then aligned to the reference genome (GRCh37/hg19) using PBmm2.

One colony of the PuBamA strain was inoculated into 20 ml of LB medium containing 50 μg/ml kanamycin and supplemented with 10 μl of toluene in a 100-ml flask with a butyl rubber cap (original culture). After a 24-h incubation at 28°C with shaking, 50 μl of the culture was inoculated into 5 ml of LB medium containing 50 μg/ml kanamycin in a 50-ml conical tube (first-passage culture). This passage was repeated twice (second- and third-passage cultures). Plasmid DNA (pPuBamA) was extracted from each culture using a typical alkaline lysis method and used as a PCR template. A DNA fragment containing the Pu promoter of the pPuBamA plasmid was amplified by PCR using the primer set IS-P-Check_F/IS-P-Check_R. The resulting amplicon from the third-passage culture was purified using the Wizard SV gel and PCR clean-up kit (Promega, Madison, WI). A sequencing library was prepared using 268 ng of the purified amplicon and a ligation sequencing kit (catalog no. SQK-LSK109; Oxford Nanopore Technologies, Oxford, UK). MinION sequencing was performed using R9.4.1 flow cells (FLO-MIN106; Oxford Nanopore Technologies). MINKNOW software v3.6.3 (Oxford Nanopore Technologies) was used for data acquisition. Real-time base calling was performed with MinIT (Oxford Nanopore Technologies) and the integrated Guppy v3.2.9 software (Oxford Nanopore Technologies) to produce fast5 and fastq files.