Nasopharyngeal 16S rRNA gene sequencing: Severe COVID-19 Patient 18
Nasopharyngeal swabs were obtained and placed in the Transport and Preservation medium (Biocomma, ShenZHEN,China). Automated total nucleic acid extraction was carried out using the NucliSENS easyMag (bioMrieux, Marcy-l'toile, France) using software version 2.1.0.1. The input volume was 500 l, and the elution volume was 50 l. Extraction was performed following the manufacturers protocol. Extracts were stored at 80C until metagenomics NGS sequencing. Extracted DNA was quantified using the Qubit Fluorometer with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). DNA libraries were prepared with the Ion 16S rRNA gene Metagenomics kit (Thermo Fisher Scientific, USA). Briefly, extracted DNA was diluted to 1.25ng/l and 12.5ng were used in total for library preparation. Amplification was performed using two sets of primers, V2-4-8 and V3-6,7-9 targeting the respective 16S rRNA gene regions, that amplify the hypervariable regions of the bacterial 16S rRNA gene locus. The two sets of reactions for each sample were combined and the DNA library was prepared using Ion Plus Fragment Library kit and Ion Xpress Barcodes Adapters according to the manufacturers protocol. The libraries were quantified by qPCR using the TaqMan Universal Library quantification kit and each library was diluted to a concentration of 100pM, amplified and template preparation was performed with the Ion One Touch System.