Qiamp extraction kit

Before freezing, dilutions of OS were plated on 1.5% nutrient agar medium (Difco) at 28 °C for 24 h. Two different morphotypes of colonies were observed. Genomic DNA of two representative isolated colonies was extracted using the QiAmp extraction kit (Qiagen) according to manufacturer recommendations. To identify bacteria, PCR amplifications of the 16S rRNA gene were performed with the following primers: 63Bis (F) 5′-GAAGAGTTTGATCATGGCTC-3′ and 153Rev (R): 5′-AAGGAGGTGATCCAGCCGCA-3′. Briefly, about 10 ng of DNA were used and amplified in a Bio-Rad thermocycler (Bio Rad, USA) with GoTaq G2 Flexi DNA Polymerase (Promega, USA) according to manufacturer recommendations. Amplification products were sequenced at MWG Eurofin center (Germany) and compared to NCBI database by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

RNA from tissues was extracted using RNeasy extraction kit (Qiagen) and RNA from BAL or nasal swabs was extracted using Qiamp extraction kit (Qiagen). SARS-CoV2 subgenomic viral RNA was quantified using primer probe sets as previously described51 and Quantifast One-Step RT-PCR master mix (Qiagen) on a QuantStudio 3 or 5 instrument (ThermoFisher). A standard curve of viral RNA of known copy number was run in parallel.

Viral RNA was extracted with the QIAmp extraction kit (Qiagen, United Kingdom) from a starting NPS specimen volume of 140 μl and final elution volume of 60 μl. Reverse transcription (RT) of RNA molecules was performed with the forward primers for each of the six amplicons. A separate RT reaction was performed for each amplicon. Typically, the 20-μl reaction mixture contained 2 μl of sample RNA. A 5-μl aliquot of the resulting cDNA was used in each 25-μl PCR mixture. The PCR mixture was incubated at 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 53°C for 30 s, and 72°C for 3.0 min and a final extension of 72°C for 10.0 min. Following PCR, aliquots of the products were run on a 0.6% agarose gel to monitor amplification success, and the products from the 6 reactions for each sample were pooled for Illumina library preparation.