# A Snapshot of the Microbiome of a Portuguese Abandoned Gold Mining Area
## 2 Materials and Methods
### Bioinformatic Data Processing  
The initial sequence data was processed at Genoinseq (Cantanhede, Portugal). The obtained raw sequencing reads were extracted from the Illumina MiSeq(r) System in fastq  
Figure 1: Sampling sites details of the (**A**) control, (**B**) tailings and (**C**) the mine. format and quality-filtered. Filtering was performed with PRINSEQ v.0.20.4 [57] and aimed to remove sequencing adapters; trimming was based on an average quality lower than 25 in a window of 5 bases and reads with less than 100 bases for the ITS2 region and 150 bases for the 16S rRNA gene were removed. Forward and reverse reads were then merged with AdapterRemoval v.2.1.5 [58] using default parameters. For bacteria, further treatment of the obtained metabarcoding data, clustering and taxonomic analyses were performed with the mothur package v.1.46.1 [59; 60]. The obtained bacterial reads were subjected to quality control measures, with chimeras being detected and removed from the datasets with the VSEARCH software v.2.16.0 [61]. The remaining high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% similarity with OptiClust [62] and then taxonomically annotated against the ARB-Silva taxonomic database v.138.1 [63] with the RDP classifier [64]. For fungi, the metabarcoding data treatment was performed with the SEED 2 bioinformatics package [65]. The obtained fungal reads were subjected to quality control measures, the enclosed ITSx software v.1.0.11 [66] was applied to extract the ITS2 sub-region (for all organisms) with default settings, chimeric sequences were detected and removed from the dataset, with the remaining reads being clustered into OTUs at 97% similarity applying the USEARCH/UPARSE (v.8.1.1861) approach [67]. Fungal OTUs were first BLAST-queried against the UNITE database v.8 [68]. Taxonomically unidentified OTUs were then further analyzed against the UNITE + INSD database in the PlutoF platform [69] through massBLASTer analysis. For both taxonomic groups, the final tables were screened to remove from the datasets OTUs assigned to non-target taxonomic groups with less than three reads. Relative abundance plots were obtained in R/R Studio v.4.1.1 with the microeco package v.0.17.0 [70] and Krona v.2.4 [71]. To evaluate individual shared OTUs between the distinct samples, Venn diagrams were constructed online with the Van de Peer Lab tool ([[http://bioinformatics.psb.ugent.be/webtools/Venn/](http://bioinformatics.psb.ugent.be/webtools/Venn/)]([http://bioinformatics.psb.ugent.be/webtools/Venn/](http://bioinformatics.psb.ugent.be/webtools/Venn/)), accessed on 1 January 2023). Complementarily, the diversity indexes and the coverage values were calculated with the mothur package v.1.46.1 [59; 60]. Additionally, the past3 statistical package v.3.20 [72] was also employed to plot a principal component analysis (PCA) based on a correlation matrix (Biplot approach) for each taxonomic group. Both datasets were independently uploaded to MicrobiomeAnalyst v.1.0 [73; 74] ([[https://www.microbiomeanalyst.ca/](https://www.microbiomeanalyst.ca/)]([https://www.microbiomeanalyst.ca/](https://www.microbiomeanalyst.ca/)), accessed on 1 January 2023) for heat map and core microbiome analysis. Since many bacterial OTUs shared a similar \"uncultured\" classification at various taxonomic levels (leading to the merging of differential taxonomic groups and possible analysis incongruences), the dataset was manually adjusted by adding the last taxonomic level identified. Hierarchical clustering heat maps were conducted reflecting Pearson distance measures with Ward clustering algorithms, and the core microbiome analysis was obtained at the family level based on a 20% sample prevalence and 0.01 relative abundance. Bacterial functional profiles were obtained with the Tax4 Fun tool v.1.0.0 [75] (for this case, using the original OTU table), while fungal functional traits were evaluated by screening the FungalTraits database v.1.2 [76] focusing mainly on primary lifestyle and decay substrate types.