Metagenomic blood units with PDI
Before nucleic acid extraction 800 l of each sample was centrifuged (17,000  g, 5 min) for removal of plasma proteins and cell debris. Consequently, a volume of 600 L of plasma was separated and treated with Turbo DNase (Turbo DNA-free kit, ThermoFisher Scientific, So Paulo, Brazil) in order to remove free host/bacterial/protozoan DNA (incubation at 37C for 30 min). This treatment removes free plasmatic DNA and the encapsulated viral nucleic acids are not degraded. After inactivation of the DNase, all of the collected 14 samples were mixed in order to form a sample pool. The total volume of the pool was extracted using the High Pure Viral Nucleic Acid Large Volume Kit (Roche, So Paulo, Brazil) following the manufacturers instructions. The extracted nucleic acids were eluted in 50 L of nuclease-free water and submitted to reverse transcription using the Superscript III First-Strand Synthesis System (ThermoFisher Scientific, So Paulo Brazil). The amplification of cDNA was performed using the QuantiTect Whole Transcriptome Kit (QIAGEN, So Paulo, Brazil). The preparation of the libraries was performed using the Nextera DNA Flex Library Preparation Kit (Illumina, San Diego, CA, USA) and the Nextera DNA CD Indexes kit following the manufacturer's instructions. The sequencing of the libraries was carried out in Illumina NextSeq 500 sequencer using the NextSeq High Output Kit v 2.5, 300 cycles (Illumina, San Diego, CA, USA) following the manufacturer's instructions.