ddRAD SNP calling
The sequences were quality assessed using FastQC (Andrews, 2010) and the
reads were demultiplexed by barcode using the
“process_radtags ” module (default parameters) of the Stacks
v1.48 bioinformatics pipeline (Catchen et al. , 2013). This module
also filtered out low-quality reads. The retained reads, now missing
variable length barcodes, were then trimmed to a standard 148 bases in
length. For each individual, matching forward and reverse reads were
then concatenated into a single longer “artificial” read using a
custom Perl script (Genbank BioProject Accession number: PRJNA1173582).
Individuals that had <100,000 reads were removed from further
processing.
The individual data were then processed using the
“denovo_map.pl ” module of stacks (-m 10 -M 2 -n 0) to assemble
and create a catalogue of genetic loci contained in the data. The Stacks
scripts “export_sql.pl ” and “populations ” and
filtering steps were used to retain all loci that fulfilled the
following criteria: a) contained exactly one SNP (in the concatenated
forward and reverse reads) to avoid physically linked markers and ensure
conserved sequence surrounding the target SNP to facilitate primer
design; b) contained exactly two alleles c) had a read depth of ≥10
reads per individual to maximize the likelihood of the SNP being real.