Microbiome DNA Extraction and Sequencing

Self-collected combined nose-throat swabs were stored at −20°C at the participants’ houses until the end of the study. After transportation on ice, samples for DNA analysis were kept at −20°C in the laboratory until further processing. Prior to DNA extraction, all samples were vortexed for 15 to 30 s, and 500 μL of the eNAT buffer was used for automatic extraction using a DNeasy 96 PowerSoil Pro QIAcube HT kit (Qiagen). Negative extraction controls were included at regular time points throughout the study. All samples were eluted with 100 μL elution buffer, and DNA concentrations were measured using the Qubit 3.0 fluorometer (Life Technologies, Ledeberg, Belgium).
Amplicon sequencing (V4 region of the 16S rRNA gene) was performed using an in-house-optimized protocol (48 (link), 49 (link)). Processing and quality control of the reads were performed using the R package DADA2, version 1.6.0, to achieve amplicon sequence variant (ASV)-level counts. Taxonomic annotation was performed using the ezbio 16S database (retrieved June 2018). All data handling and visualization were performed in R, version 3.4.4, using the tidyverse set of packages and the in-house package tidyamplicons, version 0.2.1 (publicly available at https://github.com/SWittouck/tidyamplicons), as described previously (48 (link)).