Genome sequencing and assembly of the ICBM5 carrier host strain via Nanopore

Phenol–chloroform-extracted DNA was used to prepare a sequencing library using the Rapid Barcoding Kit, according to the manufacturer’s instructions (Oxford Nanopore Technologies). The sequencing was performed on a MINION Flow Cell (R9.4.1), controlled by the MinIon software.

After sequencing, the quality control was performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (fastqc -t 16 $input‘01_reads.fastq’ -o $Step2_out—nano). Adapters were removed using PoreChop (https://github.com/rrwick/Porechop) (porechop -i $input‘01_reads.fastq’ -t 16 -v 2 -o $output‘03_reads_trimmed.fastq’ > $output‘03_porechop.log’). Assembly was performed using a set of tools from the pomoxis suite (https://github.com/nanoporetech/pomoxis). Minimap2 (minimap2 -x ava-ont -t $threads $output‘01_reads.fastq’ $output‘01_reads.fastq’ | gzip −1 > $mapping‘08_mapping.paf.gz’) and miniasm (miniasm -f $output‘01_reads.fastq’ $mapping‘08_mapping.paf.gz’ > $mapping‘08_miniasm_reads.gfa’) were used to map the .fastq files onto each other to find overlaps. Afterward, the .gfa file was converted to .fasta (awk ‘$1 ∼/S/ {print “>” $2”\ n”$3}’ $mapping‘08_miniasm_reads.gfa’ > $assembly‘09_miniasm_reads.fasta’). Assembly was done by minimap2 (minimap2 $assembly‘09_miniasm_reads.fasta’ $output‘01_reads.fastq’ > $assembly‘09_minimap_reads.paf’) and polishing was done by racon (racon -t $threads $output‘01_reads.fastq’ $assembly‘09_minimap_reads.paf’ $assembly‘09_miniasm_reads.fasta’ > $polished‘10_racon.fasta’). A last quality control was performed by quast (quast.py -t $threads -o $Step10_QC $polished‘10_racon.fasta’ $assembly‘09_miniasm_reads.fasta’) (Gurevich et al. 2013).