DOI: 10.5061/dryad.q573n5tk4
title: Methylation and gene expression data from: Differential DNA methylation across environments has no effect on gene expression in the eastern oyster

description: 1. It has been hypothesized that environmentally induced changes to gene
 body methylation could facilitate adaptive transgenerational responses to
 changing environments. 2. We compared patterns of global gene expression
 (Tag-seq) and gene body methylation (reduced representation
 bisulfite sequencing) in 80 eastern oysters (Crassostrea virginica) from
 six full-sib families, common gardened for 14 months at two sites in the
 northern Gulf of Mexico that differed in mean salinity. 3. At the time of
 sampling, oysters from the two sites differed in mass by 60% and in
 parasite loads by nearly two orders of magnitude. They also differentially
 expressed 35% of measured transcripts. However, we observed differential
 methylation at only 1.4% of potentially methylated loci in comparisons
 between individuals from these different environments, and little
 correspondence between differential methylation and differential gene
 expression. 4. Instead, methylation patterns were largely driven by
 genetic differences among families, with a PERMANOVA analysis indicating
 nearly a two orders of magnitude greater number of genes differentially
 methylated between families than between environments. 5. An analysis of
 CpG observed/expected values (CpG O/E ) across the C. virginica genome
 showed a distinct bimodal distribution, with genes from the first cluster
 showing the lower CpG O/E values, greater methylation, and higher, and
 more stable gene expression, while genes from the second cluster showed
 lower methylation, and lower and more variable gene expression. 6. Taken
 together, the differential methylation results suggest that only a small
 portion of the C. virginica genome is affected by environmentally induced
 changes in methylation. At this point, there is little evidence to suggest
 that environmentally induced m­­­ethylation states would play a leading
 role in regulating gene expression responses to new environments.
description: In May 2016, adult oysters (<i>C. virginica</i>) were
 collected by dredging from Sister Lake, LA (29˚14’57” N, 90˚55’16” W).
 These oysters were transported to the Louisiana Department of Wildlife and
 Fisheries Michael C. Voisin Oyster Hatchery in Grand Isle, LA
 (29°14'20.3" N, 90°00'11.2" W) and placed into
 off-bottom mesh cages for long-term acclimation. In October 2016, after
 six months of acclimation, the oysters were spawned at the MCV oyster
 hatchery using 3 males and 2 females. Oyster spat were reared in an
 upwelling system, individually tagged, and outplanted in one of three
 adjustable long-line mesh bags at both the Grand Isle Hatchery farm and
 near the Louisiana Universities Marine Consortium (LUMCON)
 (29°15'12.6" N, 90°39'45.9" W) on February 20th, 2017.
 Because larvae from all six families were combined for culturing,
 parentage assignments were unknown at the time of outplant, and as such
 families were unequally outplanted between sites. Oysters within each bag
 were monitored for mortality and cleaned of epibionts approximately every
 3 months over a 14-month period. On April 24th, 2018, after 14-months at
 the two outplant sites, 40 individuals were haphazardly chosen from each
 site. Shell height of each individual was measured from shell umbo to
 distal edge using a digital caliper (ABS Coolant Proof Calipers, Mituyoto
 Corporation, Japan). Approximately 1 cm<sup>2</sup> piece of
 gill tissue was sampled in the field from each individual and preserved
 with either Invitrogen RNAlater (gene expression) or 95% ethanol (DNA
 methylation). The remaining whole animal was placed in a pre-weighed 50 ml
 test tube and used to measure wet meat weight and <i>Perkinsus
 marinus</i> infection intensities. <i>Gene
 expression</i> Total RNA was extracted using a
 E.Z.N.A.® Total RNA Kit I (Omega BIO-TEK Inc., Norcross, GA, USA)
 following the manufacturer's instructions. The yield and quantity
 were initially assessed using a NanoDrop 2000 spectrophotometer. Total RNA
 extracted from the 80 individuals was sent to the University of Texas at
 Austin’s Genomic Sequencing and Analysis Facility where RNA quality
 control was confirmed using a 2100 Agilent Bioanalyzer on a Eukaryote
 Total RNA Nano chip and libraries were produced using the Tag-Sequencing
 approach (Meyer, Aglyamova, & Matz, 2011). The resulting 80
 libraries were sequenced on two lanes of an Illumina HiSeq 2500 platform,
 with 100 base pair single-end reads. Sequencing reads
 were trimmed of adapter sequences using Trimmomatic (version 0.39)
 (Bolger, Lohse, & Usadel, 2014) and base pairs with quality scores
 below 30 were removed (Table S2). The trimmed reads were mapped to the
 <i>C. virginica</i> reference genome (Gómez-Chiarri, Warren,
 Guo, & Proestou, 2015) with known haplotigs removed
 (https://github.com/jpuritz/OysterGenomeProject/tree/master/Haplotig_Masked_Genome) using the single pass option for STAR RNA-seq aligner (version 2.6.0a) (Dobin et al., 2013). Reads were mapped to gene features with the options (--quantMode GeneCounts --outFilterScoreMinOverLread 0.50 --outFilterMatchNminOverLread 0.50) specified to adjust for poly-A tail contamination. A count matrix was generated from the ReadsPerGene.out.tab output from STAR.
description: The R scripts files will provide data analysis steps needed for
 reproducing the results.