2.2. Sequencing
A restriction site-associated DNA library was generated by GBS method
(Elshire et al., 2011). To select for the best enzyme with reproducible
genomic fragments across samples (a critical criterion for GBS
experiments), we first generated a pilot library (four samples) with
Illumina MiSeq (150 bp), using two common restriction enzymes, Pstl and
ApekI. PstI provided the best reproducibility and number of variants and
was therefore chosen to generate the final library.
Genomic DNA was digested with the PstI, fragments were tagged with
individual barcodes, PCR-amplified, multiplexed, and sequenced on dual
lanes on an Illumina HiSeq 2500 platform (2x100 bp). Raw read sequences
were demultiplexed and quality checked using FastQC v0.10.1 (Wingett and
Andrews, 2018); available athttps://www.bioinformatics.babraham.ac.uk/proj‐ects/fastqc/).
Additionally, raw RADseq genomic data of P. lilfordi generated on
a previous study (Bassitta et al., 2021) were downloaded from the online
repository (PRJNA645796, 91 samples from 10 populations). The GBS
dataset (this study) and the RADseq dataset (Bassitta et al., 2021)
shared the restriction enzyme PstI, which allowed data integration for
comparative purposes (see below). Two localities of Colom and Aire were
sampled and sequenced in both studies (different individuals) (Table 1
and S1).