Data Analysis
We demultiplexed runs and conducted quality control with Torrent Suite
version 4.4.3. We retained bases above the AQ20 confidence threshold. We
trimmed sequences to 100 bp targeting the higher quality sequence at the
5’ end. We performed a de novo assembly and constructed a
pseudo-reference using Geneious Prime v. 2022.1.1 (Dotmatics). We mapped
individual sequences with BWA Galaxy Version 0.7.17.4 (Li and Durbin
2009, 2010). We used featureCounts Galaxy Version 1.6.4+galaxy1 (Liao et
al. 2013) to determine read counts of fragments within 100 bp bins
spanning the pseudo-reference. The 100 bp bins were used to count
fragments among individuals ultimately to represent variation in DNA
methylation among the CCGG sites screened. For a fragment to be
sequenced, it had to have a non-methylated CCGG site. Counting matches
to the bins across the pseudo-reference equates to variation in DNA
methylation among the CCGG sites. As epiRADseq generates data with the
zero read count result indicating DNA methylation, we used two
approaches to control for sequencing coverage differences. First, we
only analyzed individuals with 5,000 sequencing reads or higher. Second,
we standardized all statistics by sequence read count at the individual
sample level.
We used edgeR, Galaxy Version 3.24.1+galaxy1 (Robinson et al. 2010), to
detect differently methylated regions (DMR), between the 0- and 8-hour
samples, with a False Discovery Rate (FDR) of 0.05. We first compared
all samples between 0- and 8-hour; we then repeated the comparison
separately for individuals from native and introduced populations. We
determined the number of DMRs in each comparison and identified DMR that
were shared or unique to a particular comparison.
For every house sparrow, we calculated the change in DNA methylation
between the 0- and 8-hour sample for all bins with significant
differences as identified by the EdgeR analyses. We standardized each
count for each bin by sequencing depth as (observed count for bin x /
total read count) x 1,000. We compared methylation estimates among
introduced and native birds using t-tests , f-tests , and
Pearson’s correlations. Statistical tests used alpha = 0.05 and were
corrected by the sequential Bonferroni method when appropriate (Rice
1989).