DOI: 10.5061/dryad.qjq2bvqs2
title: Data from: Simulated infection induced changes in DNA methylation differ between introduced and native house sparrow (Passer domesticus)

description: As DNA methylation can change within individuals over time and regulate
 gene expression, it is important in many aspects of avian biology. It
 likely plays a critical role in the response of individuals to various
 stressors, such as infection, environmental change, and the myriad of
 novel conditions associated with introductions. Here, we use epiRADseq to
 investigate changes in DNA methylation within-individual birds over eight
 hours in response to simulated infection. We contrast house sparrows from
 introduced locations with individuals from native locations, comparing the
 number of genomic locations that change, their magnitude of change, and
 the variance among individuals of the change. We detected that introduced
 individuals change their DNA methylation at more genomic locations, with
 greater magnitude, and higher variance, compared to native individuals.
 Together, these findings support a critical role of DNA methylation in an
 individual’s response to infection, which introduces individuals likely to
 adopt an “invader phenotype,” differentiating their response from native
 individuals, and that the overall pattern of change in DNA methylation is
 congruent with epigenetic buffering.
description: We used epiRADseq to screen variation in DNA methylation among
 house sparrows on the Ion Torrent PGM platform. We followed a
 genotype-by-sequencing (GBS) protocol developed for the Ion Torrent
 platform, substituting the DNA methylation-sensitive restriction enzyme
 <em>HpaII</em> for <em>MspI</em> to construct the
 epiRADseq library. After restriction digestion, we ligated Ion Torrent
 IonXpress barcoded adaptors and y-adapters. We ran emulsion polymerase
 chain reactions following the manufacturers protocols of the Ion
 PGM-Hi-Q-View OT2-200 kit on the Ion Express OneTouch2 platform. We
 sequenced resultant fragments following manufacturers protocols of the Ion
 PGM-Hi-Q-View Sequencing 200 Kit using an Ion 316v2 BC Chips. 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 <em>de novo</em> assembly and
 constructed a pseudo-reference using Geneious Prime v. 2022.1.1. We mapped
 individual sequences with BWA Galaxy Version 0.7.17.4. We used
 featureCounts Galaxy Version 1.6.4+galaxy1 to determine read counts of
 fragments within 100 bp bins spanning the pseudo-reference. We present all
 count files, all significant count files post edgeR analysis, and summary
 information for each individual.
description: # Simulated infection induced changes in DNA methylation differ between
 introduced and native house sparrow (Passer domesticus)
 [https://doi.org/10.5061/dryad.qjq2bvqs2](https://doi.org/10.5061/dryad.qjq2bvqs2) ## Description of the data and file structure House sparrows were collected from four locations in their native range: Israel (n = 6), Norway (n = 6), Spain (n = 6), and Vietnam (n = 6), and three locations in their introduced range: Australia (n = 3), Canada (n = 6), Senegal (n = 6; Table 1). Upon capture, we took a 50 µl blood sample from the brachial vein of each bird. Immediately after this, we injected each bird with 100 µl of 1 mg/ml-1 lipopolysaccharide in sterile saline subcutaneously over the breast muscle. Post injection, we housed birds individually in wire songbird cages with food and water *ad libitum*. Eight hours post-injection, we took an additional 10 µl of blood from the brachial vein. We extracted DNA samples ending with paired 0- and 8-hour samples for each individual. We used epiRADseq to screen variation in DNA methylation among house sparrows on the Ion Torrent PGM platform. We followed a genotype-by-sequencing protocol developed for the Ion Torrent platform, substituting the DNA methylation sensitive restriction enzyme *HpaII* for *MspI* to construct the epiRADseq library. After restriction digestion, we ligated Ion Torrent IonXpress barcoded adaptors and y-adapters. We ran emulsion polymerase chain reactions following manufacturers protocols of the Ion PGM-Hi-Q-View OT2-200 kit on the Ion Express OneTouch2 platform. We sequenced resultant fragments following manufacturers protocols of the Ion PGM-Hi-Q-View Sequencing 200 Kit using an Ion 316v2 BC Chips.We demultiplexed runs and conducted quality control with Torrent Suite version 4.4.3. W e 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. We mapped individual sequences with BWA Galaxy Version 0.7.17.4. We used featureCounts Galaxy Version 1.6.4+galaxy1 to determine read counts of fragments within 100 bp bins spanning the pseudo-reference. We used edgeR, Galaxy Version 3.24.1+galaxy1, to detect differently methylated regions, between the 0- and 8-hour samples, with a False Discovery Rate of 0.05. We first compared all samples between 0- and 8-hour; we then repeated the comparison separately for native and introduced individuals. 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. ### Files and variables #### File: HOSP_0vs8hr_READ_COUNTS.xlsx **Description:**  contains summary data on read count depth for each individual in the study. Individuals are given a unique identifier that has collection location, 00 or 08 hour, and designator. ##### Variables * Read count number for each individual #### File: HOSP_DNAme_0vs8hr_ALL_DATA.xlsx **Description:** contains count data to 100bp bins for each individual in the study. Individuals are given a unique identifier that has collection location, 00 or 08 hour, and designator. All mapped loci are presented. ##### Variables * Feature count for each of the 100bp bins assigned to the pseudo reference for each individual #### File: HOSP_0vs8hr_Significant_Loci_Only.xlsx **Description:** contains count data to 100bp bins for each individual in the study. Individuals are given a unique identifier that has collection location, 00 or 08 hour, and designator. Only loci identified as containing significant differences are presented. ##### Variables * Feature count for only the significant bin of the 100bp bins assigned to the pseudo reference for each individual #### File: Additional_data_for_GLMM_and_ANOVA_analysis.csv Table 1: Correlation of Fixed Effects in a Generalized Linear Mixed Model (GLMM) This table shows the pairwise correlation coefficients between fixed effects included in a GLMM analysis. Understanding these correlations helps assess potential collinearity between predictor variables, which can affect model interpretation and stability. Table 2: Tukey HSD Post-Hoc Test for Differences in DNA Methylation Among Countries This table shows the results of Tukey’s Honestly Significant Difference (HSD) test, a post-hoc pairwise comparison method used after an ANOVA to determine which group means are significantly different from each other. Variables: Difference: The estimated mean difference in DNA methylation levels between the two countries. Lower/Upper: The bounds of the 95% confidence interval for the mean difference. Adjusted p-value: p-values corrected for multiple comparisons (Tukey adjustment). #### File: Description_of_code_used_in_the_RStudio_analysis.txt A GLMM (Gamma, log link) was used to model absolute DNA methylation change as a function of sex and native/invasive status with random intercepts for country; ANOVA followed by Tukey HSD tested for mean differences in methylation change across countries. ## Code/software * File above, Description of code used in the Studio analysis.tx, contains code used for GLMM model, ANOVA, and Tukey multiple comparisons of means analyses. - All files viewable in Microsoft Excel