Removal of the potential background contaminants determined by negative extraction controls
The susceptibility of contamination in low-biomass samples such as those used in this study, and the resulting implications for producing biologically meaningful datasets, have stimulated the development of guidelines and recommendations to mitigate the influence of contaminants in the interpretability of 16S-based microbial profiles [For the 16S datasets, the bacterial burden and the taxonomic profiles of these technical controls were subsequently used to eliminate the bias introduced by potential contaminants in the BALF 16S-based microbial profiles. The quantification of the nucleic acids presented in these control samples allowed estimating the limit of detection to discern true biological signal from background noise. We established the background cut-off to 1376.61 16S DNA copies µLFor the ITS2 sequence data we proceeded as follows. We removed reads assigned only at Kingdom level (10.77% of the total reads) as well as low count ASVs (ASVs with less than 0.01% abundance across the whole dataset), resulting in a reduction from 2331 to 596 ASVs. Implementation of 