Picogreen

DNase I activity in patients’ plasma was measured by the method previously described with some modifications^{47 (link)}. Briefly, plasma samples were diluted 1:10 with PBS + 10 mM MgSO₄ supplemented with 1 µg/ml of calf thymus DNA (Sigma-Aldrich) and stained with Pico Green (Invitrogen). To allow digestion of DNA by plasma DNase I, samples were incubated for 2 h at 37 °C. The intensity of Pico Green fluorescence was measured at time points 0 h and 2 h using a microplate reader (Victor3, Perkin Elmer). Relative DNase I activity was expressed as the ratio of relative fluorescence units measured at time point 0 h and 2 h, whereby high values may reflect high DNase I activity.

DNA was extracted using MasterPure Gram positive DNA purification kit (Lucigen, Middleton, WI, USA) according to the recommended manufacture protocol. Each sequenced sample was used to prepare the sequencing library according to the Illumina 16S Metagenomic Sequencing Library protocols. The quality and quantity of the DNA sequencing library were assessed by PicoGreen (Molecular Probes, Eugene, OR, USA) and Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). The 16S rRNA genes were amplified from input genomic DNA using 16S V3-V4 primers and a subsequent limited-cycle amplification step was performed with multiplexing indices and Illumina sequencing adapters. The final products were pooled and normalized by the PicoGreen, and the size of libraries was verified using the LabChip GX HT DNA High Sensitivity Kit (PerkinElmer, Waltham, MA, USA). Finally, the libraries were sequenced using MiSeq or HiSeq 2500 platforms (Illumina, San Diego, CA, USA).

DNA samples were first evaluated using an eGel and PicoGreen fluorometry at PerkinElmer DNA Sequencing and Analyzing Services to measure the quality and quantity, respectively. DNA samples were then physically sheared to the desired size (300–1000 bp) using a Covaris (Woburn, MA, USA). E220 focused ultrasonicator. Whole-genome libraries were prepared using an Illumina (San Diego, CA, USA) TruSeq kit following the manufacturer’s instructions. Whole-genome sequencing was performed using an Illumina HiSeq2500 instrument. Raw sequencing data were converted to FASTQ format using Illumina’s BCL2FASTQ v. 1.82. Reads were aligned to human reference GRCh37 with the Burrows‒Wheeler Aligner (BWA), v. 0.6.2. (http://bio-bwa.sourceforge.net). The resulting alignment files (bam) were filtered using Samtools (http://www.htslib.org) to remove reads that mapped to the human reference. The remaining reads were then filtered to remove low-quality reads using Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic). The remaining high-quality reads were then mapped, using BWA, against various bacterial reference genomes of interest. Reads that mapped to the genomes of interest were then used for BLAST searches.