Data from: Ocean acidification induces subtle shifts in gene expression and DNA methylation in mantle tissue of the Eastern oyster (Crassostrea virginica)
Description
Early evidence suggests that DNA methylation can mediate phenotypic responses of marine calcifying species to ocean acidification (OA). Few studies, however, have explicitly studied DNA methylation in calcifying tissues through time. Here, we examined the phenotypic and molecular responses in the extrapallial fluid and mantle (fluid and tissue at the calcification site) in adult eastern oyster (Crassostrea virginica) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO2 treatments ('control', 580 μatm; 'moderate OA', 1000 μatm; 'high OA', 2800 μatm) and sampled at 6 time points (24 hours - 80 days). We found that high OA initially induced an increase in the pH of the extrapallial fluid (pHEPF) relative to the external seawater that peaked at day 9, but then diminished over time. Calcification rates were significantly lower in the high OA treatment compared to the other treatments. To explore how oysters regulate their extrapallial fluid, gene expression and DNA methylation were examined in the mantle-edge tissue of oysters from days 9 and 80 in the control and high OA treatments. Mantle tissue mounted a significant global molecular response (both in the transcriptome and methylome) to OA that shifted through time. Although we did not find individual genes that were significantly differentially expressed under OA, the pHEPF was significantly correlated with the eigengene expression of several co-expressed gene clusters. A small number of OA-induced differentially methylated loci were discovered, which corresponded with a weak association between OA-induced changes in genome-wide gene body DNA methylation and gene expression. Gene body methylation, however, was not significantly correlated with the eigengene expression of pHEPF-correlated gene clusters. These results suggest that OA induces a subtle response in a large number of genes in C. virginica, but also indicate that plasticity at the molecular level may be limited. Our study highlights the need to reassess our understanding of tissue-specific molecular responses in marine calcifiers, as well as the role of DNA methylation and gene expression in mediating physiological and biomineralization responses to OA.
Notes
Note 1: For a complete step-by-step description of the RNA and DNA Methylation QC, mapping, and quantification pipelines see the manuscript github repository - `https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse`.
Note 2: Using scripts will require modifying data pathways. Statistical analyses use relative paths that correspond to the manuscript github repository - `https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse`.
Note 3: Raw sequence files corresponding to this study are located on NCBI with the BioProject ID - PRJNA594029. Reference files and annotations based on NCBI C. virginica genome files (002022765.2_C_virginica-3.0).
Note 4: Additional intermediate products can be found on the github repository - `https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse`.
FilesSeawater Chemistry
- `/AE17_WaterChemistry _carbChem.csv` : Biweekly water samples (Total Carbon and Alkalinity) and carbonate chemistry calculations using CO2Sys.
- `/AE17_WaterChemistry _carbChem.xlsx`: Biweekly water samples (Total Carbon and Alkalinity) and carbonate chemistry calculations using CO2Sys.
- `/AE17_WaterChemistry_weekly.csv` : Triweekly water samples of `Temperature`, `Salinity`, and `pH`.
- `/AE17_WaterChemistry_weekly.xlsx` : Triweekly water samples of `Temperature`, `Salinity`, and `pH`. Phenotypes `/AE17_metadata_ExperimentalExposureSamples_Simple.xlsx` : Contains sample meta data (treatment, time sampled, etc).
- `/AE17_metadata_ExperimentalExposureSamples_Simple.csv` : Contains sample meta data (treatment, time sampled, etc).
- `/AE17_CalcificationInfo_ExperimentalExposureSamples_Simple.xlsx` : Contains calculated calcification data.
- `/AE17_CalcificationInfo_ExperimentalExposureSamples_Simple.csv` : Contains calculated calcification data.
- `/AE17_CalcificationComplete.csv` : Calcification with associated water chemistry data generated with `/AE17_mergePhenotypeAndSW.R`.
- `/AE17_EPFpHData_ExperimentalExposureSamples_Simple.xlsx` : Contains pH measurements of extra-pallial fluid.
- `/AE17_EPFpHData_ExperimentalExposureSamples_Simple.csv` : Contains pH measurements of extra-pallial fluid.
- `/AE17_EPFpHComplete.csv` : EPF pH measurements with associated water chemistry and calculated delta pH generated with `AE17_mergePhenotypeAndSW.R`.
RNA-Seq
- `/RSEM_gene_Summary.Rdata` : RSEM transcript quantification `RData` file (all counts).
- `/RSEM_gene_EstCount.csv` : Estimated counts from RSEM.
- `/RSEM_gene_FPKM.csv` : FPKM counts from RSEM.
- `/RNAseq/RSEM_gene_TPM.csv` : TPM counts from RSEM.
- `/Target_BiomineralizationGenes.csv` : Target list of biomineralization genes.
- `/RNA_gene_postVoomAndNormalization_DGEListObj.RData`: Normalized gene count matrix (edgR and Voom pipeline).
- `/RNA_Limma_Expression_Data_forWGCNA.RData`: WGCNA expression object (Figure 7).
- `/RNA_Limma_Expression_networkConstruction_WGCNA.RData`: WGCNA network object (Figure 7).
MBD-BSseq
- `/methylKitObj_cov5Filtered_medianNormalized_united.RData` : MBD-BSseq summary counts in RData file (all counts).
- `/countMatrix_cov5Filtered_medianNormalized_methylCCounts.csv` : Methylated Cytosines counts.
- `/countMatrix_cov5Filtered_medianNormalized_totalCounts.csv` : Total Cytosines counts (coverage).
- `/countMatrix_cov5Filtered_medianNormalized_unMethylCCounts.csv` : Unmethylated Cytosines counts.
- `/countMatrix_cov5Filtered_medianNormalized_beta.tar.xz` : DNA Methylation proportion, beta (compressed).
- `/CpGCoordinates.csv` : List of CpG genomic coordinates.
- `/20200202_geneBeta_cov5_byFeature.RData`: Beta values per gene.
- `/DNAm_methylkit_dm50_q01_sigSummaryTable.csv`: Differentially methylated loci (DML).
References
- `/CDS_wGoTerms_GeneLOC.RData`: Coding regions with full GO term descriptions.
- `/STAR_gnomon_tximportGeneFile.RData`: Gene description file.
Scripts
(see https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse for additional notes on using these scripts)
-
RNA-Seq Pipeline
- `02_STAR_genomeCreate.sh` : Script for creating index for STAR mapping.
- `03A_STAR_1Pass.sh` : Script 1st pass of STAR mapping.
- `03B_STAR_2Pass.sh` : Script of 2nd pass of STAR mapping.
- `04A_RSEM_createRefFromStar.sh` Script for creating genome reference for RSEM quantification.
- `04B_RSEM_calcExp.sh` : Script for RSEM quantification.
- `05_filtering_CreatingDGEListObj.R` : Script for creating standardized DGEList object of RNAseq data.
- `06_CreatingWGCNAObj.R` : Script for performing WGCNA clustering and object creation.
-
MBD-BSseq Pipeline
- `00_concateRename.R` : Used to merge data from sequence facility (not needed when downloading files from NCBI).
- `01_seqQualityTrim.sh` : Script for performing QC and trimming step on raw reads.
- `02_bismarkMapping.sh` : Script for performing mapping using bismark.
- `03_cytosineReport.sh` : Script for generating cytosine reports (estimates of methylation for all CpGs) for each individual.
- `04_methylationMatrix_diffMethylation_methylKit.R` : Script for generation methylation matrices and differential methylation analyses using MehtylKit.
- `05A_CpGIntersectionByFeature.sh` : Script for intersecting CpGs by genomic feature.
- `05B_CpGCountByFeature.sh`: Script for counting CpGs per genomic feature.
-
Statistics Scripts
- `AE17_diffExpression.R` : Script for performing differential expression using DGEList object.
- `AE17_fig1_EPFtimeseries.R` : Script for analyzing complete EPF pH timeseries data (Figure 1).
- `AE17_fig2_EPFfinal_calcification.R` : Script for analyzing long-term EPF pH and calcification data (Figure 2).
- `AE17_fig3_DNAm.R` : Script for performing PERMANOVA, dapc, and other genome-wide analyses with the MBD-BSseq data (Figure 3).
- `AE17_fig4_geneExpression.R` : Script for performing PERMANOVA, dapc, and other genome-wide analyses with the RNA-seq data (Figure 4).
- `AE17_fig5_DNAmvsGE.R` : Script for examining association between DNA Methylation and gene expression data (Figure 5).
- `AE17_fig6_diffDNAmvsGE.R` : Script for evaluation association between differential gene expression and difference in DNA methylation (Figure 6).
- `AE17_WGCNAmultiComp.R` : Script for examining correlation between gene body DNA methylation, gene cluster expression, and EPF pH (Figure 7).
-
Additional
- `AE17_mergePhenotypeAndSW.R` : Script used to merge meta data (`/AE17_metadata_ExperimentalExposureSamples_Simple.csv`), water chem data (`/AE17_WaterChemistry_weekly.csv`) and phenotype data (`/AE17_EPFpHData_ExperimentalExposureSamples_Simple.csv`;`/AE17_CalcificationInfo_ExperimentalExposureSamples_Simple.csv`) and create merged table (`/AE17_EPFpHComplete.csv`;`/AE17_CalcificationComplete.csv`).
- `basicR_functions.R` : List of simple functions used in other scripts.
Funding provided by: National Science Foundation
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000001
Award Number: BIO-OCE 1635423
Funding provided by: National Science Foundation FSML Program*
Crossref Funder Registry ID:
Award Number: DBI 1722553
Funding provided by: National Science Foundation FSML Program
Crossref Funder Registry ID:
Award Number: DBI 1722553
Files
AE17_CalcificationComplete_revised.csv
Files
(426.2 MB)
| Name | Size | Download all |
|---|---|---|
|
md5:19b775ea0f78100aff46cd189a99ed0f
|
999 Bytes | Download |
|
md5:6e5f4ba756567e7630f6abf6d0c8bcb2
|
976 Bytes | Download |
|
md5:ff1f74ed24a34a77e7bd226d93f250a9
|
1.5 kB | Download |
|
md5:c226070c020a1f7020bcfd24fc8ce89e
|
646 Bytes | Download |
|
md5:5647829d0033c4ce250943e65448db90
|
1.0 kB | Download |
|
md5:c1e522e6a58bc40ca04c8955053d94e6
|
1.5 kB | Download |
|
md5:c5127ec3f2f25e302ba1100f8436fc93
|
1.9 kB | Download |
|
md5:a4662be5cec18d9425a152831f47b7e7
|
29.2 kB | Download |
|
md5:72faec867d32b03582485d4774195211
|
333 Bytes | Download |
|
md5:8ae5053612afaad3e8435582c17aa51f
|
935 Bytes | Download |
|
md5:783f5dde79ce6e9273ddf305911105c2
|
5.6 kB | Download |
|
md5:83607733dd56d808461416b5869b2cb0
|
8.9 kB | Download |
|
md5:2376b0d654655862e4fa7b99458b00f8
|
3.4 kB | Download |
|
md5:e6bc0c5066533ec22c13437af75004ac
|
9.2 kB | Download |
|
md5:895cfcc5b31b85d7a9a128fdd537d0de
|
26.9 MB | Download |
|
md5:b463ccab6a198493c6419680afbace4b
|
15.0 kB | Preview Download |
|
md5:df410567a313bc7c7e1a8d3579062453
|
2.3 kB | Preview Download |
|
md5:5eca1a85f1b55db50d839cb4fc2d7183
|
29.2 kB | Preview Download |
|
md5:db3439afdec1c67c2f6fd825c898a087
|
86.0 kB | Download |
|
md5:40abc428c1e3b0fde19fddac8ae2711a
|
16.4 kB | Preview Download |
|
md5:43bff96b264f7668522cce0a912b2457
|
11.1 kB | Download |
|
md5:59a1c9ce6d15324d9a00081548779fb1
|
31.6 kB | Preview Download |
|
md5:d7dc7a3ce788d9338d9403cd387fd5a3
|
7.3 kB | Preview Download |
|
md5:76e9e9ea44b249f3f351d06daf3ea3a6
|
14.2 kB | Download |
|
md5:4adc40c9b8925264d398c0b1f0626ef8
|
12.5 kB | Preview Download |
|
md5:f3cf2bc31ab568ebe7e55a096ac8e90e
|
60.0 kB | Preview Download |
|
md5:5621a848fbd9e00eb4409e8589900435
|
11.5 kB | Download |
|
md5:032fa5be9ee2e062d8e0a7a33c037416
|
9.8 kB | Download |
|
md5:602f2cd63af30e2678c723853aa774d6
|
10.7 kB | Download |
|
md5:a215cab9dbbadcfa8d2be0b655c5c5b5
|
6.6 kB | Download |
|
md5:fe412cb5d49cc65baa2188500d420a84
|
9.1 kB | Download |
|
md5:aa2f62c802204b17166dd14801f87ec4
|
7.7 kB | Download |
|
md5:4b38ec6d0547438e58a213bb596fa1c8
|
20.6 kB | Download |
|
md5:86cef56ea6356e787cc2fa63ce17c41e
|
11.8 kB | Download |
|
md5:de9c88fca756df751506819a14f7b23d
|
8.0 kB | Preview Download |
|
md5:bf981fff13f7f48ee982de33b5c6994d
|
14.9 kB | Download |
|
md5:dad8eb60f3d6f7ead15f848de9daa735
|
1.4 kB | Preview Download |
|
md5:db336025d9b9dc600cc8e81307fb7cc6
|
8.7 kB | Download |
|
md5:ce576ddf01d30eec144297906f1f7fab
|
53.3 kB | Preview Download |
|
md5:78f24b85c6f699aa609a597dbd1a5912
|
52.3 kB | Download |
|
md5:4d94343a9111ca5c174f37ee45f355d7
|
2.2 kB | Download |
|
md5:0ef4cd44c2684e224122d3d8485325d5
|
6.6 MB | Download |
|
md5:cd4873ce20a5d3b31abf884e1c067853
|
19.0 MB | Download |
|
md5:a5d4dfbc20dddeb1d1d49cb71ce5fb36
|
29.3 MB | Preview Download |
|
md5:9c42886872a8046bd60e34cfd17b0545
|
30.7 MB | Preview Download |
|
md5:3804ea62e2258e5e08e781acc13c5546
|
23.1 MB | Preview Download |
|
md5:aafe57b7d9442e35e25e5d9b6d37d8c1
|
108.7 MB | Download |
|
md5:36baf132f7ec139930ebc2be88a88da6
|
12.8 MB | Preview Download |
|
md5:291c047602f14528be8d13ac1ee2701c
|
70.6 kB | Preview Download |
|
md5:ab1b99de988fa17b0ca02a23cd1efb06
|
1.7 MB | Download |
|
md5:86d02c3b323f32a5d9fa188d7bfe82e9
|
23.5 MB | Download |
|
md5:5fae92550a3178acf80a702d7992265f
|
43.4 MB | Download |
|
md5:982595edcbf1d828191b41931d6d3e5f
|
21.7 MB | Download |
|
md5:bf33a28f1e6c34e3826aacc2ecf52357
|
21.8 MB | Download |
|
md5:18f945f6eb31c78f6eaa6f269d415d9a
|
23.4 MB | Download |
|
md5:198f1cb61fdb53084fb1a749323b908b
|
1.9 MB | Download |
|
md5:4ffcddb473d4c19e034056f952c6b492
|
6.0 MB | Download |
|
md5:6838e3c4b093a97744ed65fca32f7599
|
3.0 MB | Download |
|
md5:6c7a7e2de8d02ab0c97f6d1c4228d05f
|
361.5 kB | Download |
|
md5:19e65764a254e29839645182235ddb38
|
4.1 MB | Preview Download |
|
md5:ac90cd55d122e9dff509746fc0c20618
|
4.5 MB | Preview Download |
|
md5:6139eb23eb149136eccec76153d81f74
|
7.2 MB | Download |
|
md5:400917aaf31668183a03432a379ea4c4
|
4.6 MB | Preview Download |
|
md5:633a1ca9056e5d1c26d573a293f0eaa4
|
1.3 MB | Download |
|
md5:482cdb36fed4109a25e6e0c741c5c97e
|
15.6 kB | Preview Download |
|
md5:fed092f5e5ed1d3a60b3e29125520049
|
35.3 kB | Download |
Additional details
Related works
- Is cited by
- 10.3389/fmars.2020.566419 (DOI)
- Is source of
- https://github.com/epigeneticstoocean/AE17_Cvirginica_MolecularResponse (URL)