2.2.2 Assembly of RADseq datasets
The raw reads from the MiSeq sequencing were processed and assembled
Stacks v2.3 (Rochette et al., 2019) as described earlier for metagenomic
datasets of lichens (Alonso-García et al., 2021). In short, we
demultiplexed reads of individuals from the pool of raw sequence reads
based on their barcodes with the script ‘process-radtags’. The
demultiplexed reads of each individual were aligned to the reference
genome database using Bowtie2 (Langmead and Salzberg, 2012). The script
‘gstacks’ with default parameters was used to identify SNPs in the reads
aligned to the reference genome. The SNP data was then analyzed and
filtered with the script ‘populations’ considering each individual a
single population. The final dataset did not allow heterozygosity at a
locus (–max-obs-het 0) and was filtered for a minimum minor allele
frequency of 5% (–min-maf .05). Only the first SNP per locus
(–write-single-snp) was retained when present in at least 30% (-R
0.3) of the individuals.