Differential DNA Sequencing of Microvesicles

DNA was extracted from the isolated MV fraction using ISOPLANT (Nippon Gene) according to the manufacturer’s instructions. A Nextera XT DNA library preparation kit (Illumina) was used for paired-end libraries. DNA-seq was performed using an Illumina MiSeq^TM system.
Low-quality read removal and adapter trimming of the raw FASTQ file obtained were performed using fastp software (version 0.20.1) (Chen et al., 2018 (link)) with the “-q 20 -3” options. The cleaned FASTQ file was mapped to the P. denitrificans Pd1222 genome using Bowtie2 (version 2.4.4). Prior to mapping, a FASTA file of the genome was indexed using the “Bowtie-build” command in Bowtie2. The resulting BAM files were indexed using SAMtools (version 1.12) (Li et al., 2009 (link)). A GTF file of the P. denitrificans Pd1222 genome partitioned into 1,000-bp windows was created using R script, and the number of reads mapped to every 1,000 bp in the genome was counted using featureCounts software (version 2.0.1) (Liao et al., 2014 (link)) with the GTF file and “-p -O -M -C -f -t region -g ID -s 0” options. The number of reads mapped to the gene was also counted with the GTF file of P. denitrificans Pd1222 and the “-p -O -M -C -f -t CDS -g gene_id -s 0” options.