Optimized mNGS Viral Detection Protocol

As previously described, we used an mNGS protocol optimized in our lab [43 (link)]. Briefly, after thawing all the samples were supplemented with MS2 bacteriophage (Levivirus genus) from a commercial kit (MS2, IC1 RNA internal control; r-gene, bioMérieux) to check the validity of the process. Only the RNA internal control MS2 was added because it validates all the steps of our protocol (including RT stage during amplification) in contrast to a DNA internal control. A no-template control (NTC) consisting of RNase free water was implemented to evaluate the contamination during the process. An additional negative control consisting of viral transport medium was added. For sample viral enrichment, a 3-step method was applied to 220 μL of vortexed sample spiked with MS2 (low-speed centrifugation, followed by the filtration of the supernatant and then Turbo DNase treatment), as described in detail in Bal et al. [43 (link)]. After viral enrichment, the total nucleic acids were extracted using one of the three methods selected for the study described above. After random nucleic acid amplification using modified whole transcriptome amplification (WTA2, Sigma-Aldrich, Darmstadt, Germany), libraries were prepared using the Nextera XT DNA Library kit and sequenced with Illumina NextSeq 500 ™ using a 2 x 150 PE high-output flow cell (Illumina, San Diego, CA, USA).