Nanopore-based Plasmid Sequencing and Analysis

Although a local basecaller script was used during the run, there was still a small amount of reads that were not basecalled due to the generation of raw data in a rapid mode. Albacore basecalling software (v1.0.3) was used to generate fast5 files harboring the 1D DNA sequence from fast5 files with only raw data in the tmp folder. Also, the read_fast5_basecaller.py script in Albacore was used to de-multiplex the 12 samples from basecalled fast5 files (except the files in fail folder) based on the 12 barcodes in SQK-RBK001. The Poretools toolkit was utilized to extract all the DNA sequences from fast5 to fasta format among the 12 samples, respectively (Poretools, RRID:SCR_015879) [18 (link)]. The Canu assembly tool (v1.3; Canu, RRID:SCR_015880) [14 (link)] was used to perform de novo assembly of complete plasmid sequences based on nanopore 1D long reads in 3 consecutive stages including correction, trimming, and assembly [14 (link)]. Due to the possibility of the contamination of bacterial chromosomal DNA in the plasmid samples and the large variation of the size of the plasmids, the parameter of genomeSize was set at 0.5, 1, 2, and 4 m, respectively, to optimize the assembly results to obtain circular plasmid sequences of interest. The sizes and the numbers of plasmids determined by S1–pulsed-field gel electrophoresis (PFGE) were used to confirm the assembled results. High-quality complete plasmids were constructed by hybrid de novo assembly of Illumina short reads and nanopore long reads data using the Unicycler v0.3 tool [19 (link)]. NanoOK was adopted to evaluate the quality of nanopore long reads [20 (link)]. BWA MEM was used to align long reads against reference plasmids (BWA, RRID:SCR_010910) [21 (link)].
To assess the distribution of resistance genes, mobile elements, and replicon genes, the corresponding databases were downloaded [21 (link)–23 (link)] and BLASTN was performed among the finished plasmids (BLASTN, RRID:SCR_001598). The result was visualized by the tool Genesis [24 (link)]. Easyfig was utilized to compare the detailed structures of the MDR plasmids (Easyfig, RRID:SCR_013169) [25 (link)].