Ion Torrent S5 sequencing of gut microbiota: Mild COVID-19 Patients
Briefly, 16S DNA libraries were prepared using the Ion 16S Metagenomics Kit targeted panel (Thermo Fisher Scientific) and each sample was individually identified with the Ion Xpress Barcode Adapters Kits (Thermo Fisher Scientific). All available regions were amplified using the Ion 16S Metagenomics Kit (Thermo Fisher Scientific). Amplified fragments were then prepared for sequencing using the Ion CHEF system (Thermo Fisher Scientific) and loaded into Ion 318 Chip Kit v2 BC (Thermo Fisher Scientific). Sequencing runs were performed on an Ion S5 System (Thermo Fisher Scientific) aiming for a mean sequencing depth coverage of 12000. Sequencing depths were not normalized in order to achieve a better identification of alpha diversity in each sample. Sequencing data was filtered for length (cutadapt -m 80) and for quality (fastx_trimmer -l 280) after which the V3 and V4 regions were extracted (Mothur align.seqs and screen.seqs). The resulting fastq file was used for taxonomy.