2.2 | Data preparation
The Illumina NovaSeq sequencing yielded a total of 25 x
109 assigned 150 bp reads, and a total of 45 – 100 Gb
of sequence data for each of the 28 pooled DNA libraries. Raw DNA
sequence reads from the two separate pooled libraries per sample
location were pooled for processing purposes. Raw sequences were
processed using the Trimmomatic V0.36 program (Bolger et al.2014) by removing NexteraTM adaptors and discarding
all reads that had a Phred score below 20. All retained reads were
subsequently aligned to the H.rubra reference genome (NCBI RefSeq
QXJH00000000.1; Gan et al. 2019) using the PPalign package in the
PoolParty pipeline (Micheletti & Narum 2018) with default parameters.
Single nucleotide polymorphisms (SNPs) were called using PoolFstat
(Hivert et al. 2018) where SNPs were required to have a read
depth of 20 – 200 reads to be called. SNPs with a minor allele
frequency of ≥ 0.05 were used for downstream genomic analysis.