The Methylome in Female Adolescent Conduct Disorder: Neural Pathomechanisms and Environmental Risk Factors
Differential Methylation Analysis
AG Chiocchetti
29 Dezember 2020
Last updated: 2021-09-24
Checks: 7 0
Knit directory: femNATCD_MethSeq/
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## load Data ####
load(paste0(Home,"/output/ProcessedData.RData"))define model
# test SVAs for significant difference between groups
svas = names(colData(dds_filt))[str_detect(names(colData(dds_filt)), "^V")]
testframe = as.data.frame(colData(dds_filt)) %>% dplyr::select(group, all_of(svas))
svas_sig = c()
for (s in svas){
tmp = t.test(testframe[,s]~testframe[,"group"])$p.value
if(tmp <= 0.05){
svas_sig=c(svas_sig,s)
}
}
#add significant sva to model
sva = paste0(svas_sig, collapse = "+")
dds_filt$group <- relevel(dds_filt$group, ref = "CTRL")
tmpdes=gsub(" group", paste0(sva,"+group"), as.character(design(dds_filt))[2])
design(dds_filt) = formula(paste0("~",tmpdes))
designmatrix = model.matrix(design(dds_filt), colData(dds_filt))model formula: Epi ~ 0 + Age + int_dis + medication + contraceptives + cigday_1 + V8 + group
analyze
if (reanalyze){
dds_filt = DESeq(dds_filt)
savefile=TRUE
} else
{
if(file.exists(paste0(Home,"/output/dds_filt_analyzed.RData"))){
load(paste0(Home,"/output/dds_filt_analyzed.RData"))
savefile=FALSE
}else {
print("no preprocessed file found data is reanalyzed")
dds_filt = DESeq(dds_filt)
savefile=TRUE
}
}
res = results(dds_filt)
summary(res)
out of 216102 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up) : 0, 0%
LFC < 0 (down) : 1, 0.00046%
outliers [1] : 0, 0%
low counts [2] : 0, 0%
(mean count < 4)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?resultsres <- res %>% cbind(as.data.frame(rowRanges(dds_filt)))
res$ID = paste0("tag_",1:nrow(res), "_",res$gene)
rownames(res)=res$ID
res$CHR = as.numeric(res$seqnames)
restop = as.data.frame(res[order(res$pvalue, decreasing = F), ])
restop = restop[restop$pvalue<=10E-3,]
if(savefile){
save(dds_filt, file=paste0(Home,"/output/dds_filt_analyzed.RData"))}sumarize results linear model
topN = 10
DESeq2::plotDispEsts(dds_filt)
lp <- -log10(res$pvalue)
ord <- order(lp, decreasing = TRUE)[1:topN]
qqman::qq(res$pvalue, main="uncorrected p-value")
qqman::manhattan(res, chr="CHR", bp="start", p="pvalue", snp="ID", col = c("#366697", "#BB8300"))
ressum= res[ord,]
display_tab(ressum)| baseMean | log2FoldChange | lfcSE | stat | pvalue | padj | seqnames | start | end | width | strand | raw_gene | gene | distance | feature | cpg | tf_binding | samples_per_target | mean | std | baseMean.1 | baseVar | allZero | dispGeneEst | dispGeneIter | dispFit | dispersion | dispIter | dispOutlier | dispMAP | Age | int_disno | int_disyes | medicationyes | contraceptivesyes | cigday_1 | V8 | groupCD | SE_Age | SE_int_disno | SE_int_disyes | SE_medicationyes | SE_contraceptivesyes | SE_cigday_1 | SE_V8 | SE_groupCD | WaldStatistic_Age | WaldStatistic_int_disno | WaldStatistic_int_disyes | WaldStatistic_medicationyes | WaldStatistic_contraceptivesyes | WaldStatistic_cigday_1 | WaldStatistic_V8 | WaldStatistic_groupCD | WaldPvalue_Age | WaldPvalue_int_disno | WaldPvalue_int_disyes | WaldPvalue_medicationyes | WaldPvalue_contraceptivesyes | WaldPvalue_cigday_1 | WaldPvalue_V8 | WaldPvalue_groupCD | betaConv | betaIter | deviance | maxCooks | ID | CHR | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| tag_134778_MIR4500HG,SLITRK5 | 13.294 | -1.128 | 0.201 | -5.600 | 0 | 0.005 | chr13 | 88324376 | 88324381 | 6 |
|
MIR4500HG,SLITRK5 | MIR4500HG,SLITRK5 | 0 | upstream | cpg | Pol2//GR | 100 | 15.706 | 12.631 | 13.294 | 61.760 | FALSE | 0.177 | 11 | 0.171 | 0.176 | 11 | FALSE | 0.176 | -0.045 | 4.741 | 5.031 | 0.167 | -0.126 | 0.031 | 0.706 | -1.128 | 0.054 | 0.854 | 0.884 | 0.214 | 0.192 | 0.017 | 0.879 | 0.201 | -0.839 | 5.551 | 5.689 | 0.781 | -0.660 | 1.763 | 0.803 | -5.600 | 0.401 | 0.000 | 0.000 | 0.435 | 0.509 | 0.078 | 0.422 | 0 | TRUE | 6 | 645.105 | NA | tag_134778_MIR4500HG,SLITRK5 | 13 |
| tag_204782_PDGFB | 14.798 | 0.792 | 0.174 | 4.558 | 0 | 0.308 | chr22 | 39629982 | 39629987 | 6 |
|
PDGFB | PDGFB | 0 | intronic | NoCpg | 100 | 16.873 | 10.585 | 14.798 | 67.579 | FALSE | 0.131 | 11 | 0.154 | 0.135 | 11 | FALSE | 0.135 | 0.118 | 1.825 | 1.468 | -0.032 | -0.272 | -0.009 | 0.172 | 0.792 | 0.048 | 0.767 | 0.792 | 0.184 | 0.169 | 0.015 | 0.810 | 0.174 | 2.448 | 2.380 | 1.854 | -0.176 | -1.610 | -0.606 | 0.212 | 4.558 | 0.014 | 0.017 | 0.064 | 0.860 | 0.107 | 0.545 | 0.832 | 0 | TRUE | 6 | 650.103 | NA | tag_204782_PDGFB | 22 | |
| tag_35466_MIR4792 | 5.316 | -1.543 | 0.339 | -4.552 | 0 | 0.308 | chr3 | 24561792 | 24561797 | 6 |
|
MIR4792 | MIR4792 | 0 | downstream | NoCpg | Pol2//CCNT2//ZBTB7A_(SC-34508)//GABP//HDAC2_(SC-6296)//TAF1//Pol2-4H8//NRSF//GATA-2//ELF1_(SC-631)//GATA2_(CG2-96)//TAL1_(SC-12984) | 86 | 5.882 | 5.642 | 5.316 | 24.526 | FALSE | 0.494 | 11 | 0.420 | 0.481 | 12 | FALSE | 0.481 | -0.058 | 3.348 | 3.980 | 0.265 | 0.228 | 0.076 | 1.156 | -1.543 | 0.090 | 1.417 | 1.466 | 0.352 | 0.311 | 0.028 | 1.430 | 0.339 | -0.647 | 2.363 | 2.715 | 0.752 | 0.731 | 2.711 | 0.808 | -4.552 | 0.517 | 0.018 | 0.007 | 0.452 | 0.465 | 0.007 | 0.419 | 0 | TRUE | 6 | 525.501 | NA | tag_35466_MIR4792 | 3 |
| tag_120467_PVRL1 | 36.541 | -0.468 | 0.103 | -4.538 | 0 | 0.308 | chr11 | 119544539 | 119544544 | 6 |
|
PVRL1 | PVRL1 | 0 | intronic | NoCpg | Pol2-4H8//NFKB//BATF//YY1_(C-20)//CTCF//TCF12//CHD2_(N-1250)//RXRA//Pol2//SMC3_(ab9263) | 102 | 42.863 | 26.350 | 36.541 | 118.993 | FALSE | 0.038 | 7 | 0.065 | 0.042 | 11 | FALSE | 0.042 | -0.030 | 5.753 | 5.685 | 0.303 | 0.117 | 0.016 | -0.529 | -0.468 | 0.028 | 0.444 | 0.459 | 0.109 | 0.099 | 0.009 | 0.461 | 0.103 | -1.066 | 12.948 | 12.392 | 2.774 | 1.178 | 1.746 | -1.148 | -4.538 | 0.287 | 0.000 | 0.000 | 0.006 | 0.239 | 0.081 | 0.251 | 0 | TRUE | 4 | 725.887 | NA | tag_120467_PVRL1 | 11 |
| tag_137745_KLHDC2 | 18.591 | -0.686 | 0.154 | -4.447 | 0 | 0.321 | chr14 | 50234989 | 50234994 | 6 |
|
KLHDC2 | KLHDC2 | 0 | UTR5 | cpg | Pol2//TAF1//TBP//CCNT2//Sin3Ak-20//HMGN3//Pol2-4H8//HEY1//Oct-2 | 102 | 22.373 | 17.109 | 18.591 | 61.296 | FALSE | 0.093 | 8 | 0.123 | 0.098 | 11 | FALSE | 0.098 | -0.028 | 4.755 | 4.974 | 0.334 | -0.145 | 0.039 | 0.049 | -0.686 | 0.042 | 0.659 | 0.682 | 0.162 | 0.147 | 0.013 | 0.679 | 0.154 | -0.677 | 7.211 | 7.297 | 2.066 | -0.987 | 2.979 | 0.072 | -4.447 | 0.498 | 0.000 | 0.000 | 0.039 | 0.324 | 0.003 | 0.943 | 0 | TRUE | 4 | 667.003 | NA | tag_137745_KLHDC2 | 14 |
| tag_197061_BMP7 | 9.825 | 1.043 | 0.235 | 4.442 | 0 | 0.321 | chr20 | 55812238 | 55812243 | 6 |
|
BMP7 | BMP7 | 0 | intronic | NoCpg | 96 | 10.961 | 8.664 | 9.825 | 93.487 | FALSE | 0.272 | 12 | 0.229 | 0.264 | 12 | FALSE | 0.264 | 0.011 | 2.921 | 2.234 | -0.190 | -0.073 | -0.031 | 0.077 | 1.043 | 0.065 | 1.034 | 1.069 | 0.250 | 0.229 | 0.021 | 1.102 | 0.235 | 0.165 | 2.825 | 2.091 | -0.761 | -0.318 | -1.521 | 0.070 | 4.442 | 0.869 | 0.005 | 0.037 | 0.447 | 0.751 | 0.128 | 0.944 | 0 | TRUE | 6 | 614.616 | NA | tag_197061_BMP7 | 20 | |
| tag_68218_BEND3 | 8.151 | -0.968 | 0.226 | -4.280 | 0 | 0.479 | chr6 | 107436782 | 107436787 | 6 |
|
BEND3 | BEND3 | 0 | upstream | cpg | E2F1//Pol2//HA-E2F1//AP-2gamma//Egr-1//E2F6_(H-50)//Pol2-4H8 | 98 | 9.745 | 7.996 | 8.151 | 24.042 | FALSE | 0.179 | 8 | 0.275 | 0.195 | 11 | FALSE | 0.195 | -0.060 | 4.147 | 4.160 | 0.356 | 0.056 | 0.048 | -1.633 | -0.968 | 0.060 | 0.955 | 0.987 | 0.239 | 0.214 | 0.019 | 0.943 | 0.226 | -0.985 | 4.342 | 4.217 | 1.494 | 0.262 | 2.480 | -1.733 | -4.280 | 0.324 | 0.000 | 0.000 | 0.135 | 0.793 | 0.013 | 0.083 | 0 | TRUE | 5 | 563.376 | NA | tag_68218_BEND3 | 6 |
| tag_10516_C1orf52 | 6.161 | -1.091 | 0.255 | -4.278 | 0 | 0.479 | chr1 | 85722202 | 85722207 | 6 |
|
C1orf52 | C1orf52 | 0 | intronic | NoCpg | 92 | 7.520 | 6.511 | 6.161 | 19.477 | FALSE | 0.210 | 10 | 0.363 | 0.239 | 9 | FALSE | 0.239 | -0.088 | 4.098 | 4.510 | 0.592 | -0.160 | 0.064 | -0.449 | -1.091 | 0.068 | 1.071 | 1.107 | 0.263 | 0.241 | 0.021 | 1.092 | 0.255 | -1.299 | 3.827 | 4.075 | 2.253 | -0.667 | 3.001 | -0.411 | -4.278 | 0.194 | 0.000 | 0.000 | 0.024 | 0.505 | 0.003 | 0.681 | 0 | TRUE | 6 | 529.701 | NA | tag_10516_C1orf52 | 1 | |
| tag_15433_METTL13 | 9.501 | -0.858 | 0.202 | -4.251 | 0 | 0.479 | chr1 | 171751562 | 171751567 | 6 |
|
METTL13 | METTL13 | 0 | intronic | NoCpg | 100 | 11.255 | 9.282 | 9.501 | 30.942 | FALSE | 0.137 | 11 | 0.237 | 0.154 | 11 | FALSE | 0.154 | -0.019 | 3.838 | 3.822 | 0.235 | 0.085 | 0.001 | -0.378 | -0.858 | 0.054 | 0.859 | 0.888 | 0.217 | 0.194 | 0.018 | 0.901 | 0.202 | -0.346 | 4.471 | 4.307 | 1.084 | 0.436 | 0.046 | -0.420 | -4.251 | 0.730 | 0.000 | 0.000 | 0.278 | 0.663 | 0.964 | 0.674 | 0 | TRUE | 4 | 575.449 | NA | tag_15433_METTL13 | 1 | |
| tag_55541_ANKRD34B | 7.256 | 1.048 | 0.247 | 4.240 | 0 | 0.479 | chr5 | 79855772 | 79855777 | 6 |
|
ANKRD34B | ANKRD34B | 0 | exonic | NoCpg | 92 | 8.422 | 6.364 | 7.256 | 28.876 | FALSE | 0.262 | 8 | 0.309 | 0.271 | 11 | FALSE | 0.271 | 0.154 | 0.071 | 0.263 | -0.411 | 0.037 | -0.076 | -2.467 | 1.048 | 0.069 | 1.100 | 1.136 | 0.265 | 0.239 | 0.022 | 1.105 | 0.247 | 2.223 | 0.065 | 0.232 | -1.551 | 0.153 | -3.434 | -2.233 | 4.240 | 0.026 | 0.948 | 0.817 | 0.121 | 0.878 | 0.001 | 0.026 | 0 | TRUE | 6 | 562.754 | NA | tag_55541_ANKRD34B | 5 |
write.csv(res, file = paste0(Home,"/output/LME_Results.csv"), row.names = T)
write.csv2(restop, file = paste0(Home,"/output/LME_Results_Sig.csv"), row.names = T)EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'pvalue',
pCutoff = 10e-3,
FCcutoff = 1)
sessionInfo()R version 4.0.3 (2020-10-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)
Matrix products: default
locale:
[1] LC_COLLATE=German_Germany.1252 LC_CTYPE=German_Germany.1252
[3] LC_MONETARY=German_Germany.1252 LC_NUMERIC=C
[5] LC_TIME=German_Germany.1252
attached base packages:
[1] parallel stats4 stats graphics grDevices utils datasets
[8] methods base
other attached packages:
[1] scales_1.1.1 compareGroups_4.4.6
[3] kableExtra_1.3.1 qqman_0.1.4
[5] EnhancedVolcano_1.8.0 ggrepel_0.9.1
[7] RCircos_1.2.1 doParallel_1.0.16
[9] iterators_1.0.13 foreach_1.5.1
[11] DESeq2_1.30.0 SummarizedExperiment_1.20.0
[13] Biobase_2.50.0 MatrixGenerics_1.2.0
[15] matrixStats_0.57.0 GenomicRanges_1.42.0
[17] GenomeInfoDb_1.26.2 IRanges_2.24.1
[19] S4Vectors_0.28.1 BiocGenerics_0.36.0
[21] forcats_0.5.0 stringr_1.4.0
[23] dplyr_1.0.2 purrr_0.3.4
[25] readr_1.4.0 tidyr_1.1.2
[27] tibble_3.0.4 ggplot2_3.3.3
[29] tidyverse_1.3.0 RColorBrewer_1.1-2
[31] knitr_1.30 workflowr_1.6.2
loaded via a namespace (and not attached):
[1] uuid_0.1-4 readxl_1.3.1 backports_1.2.0
[4] systemfonts_0.3.2 splines_4.0.3 BiocParallel_1.24.1
[7] digest_0.6.27 htmltools_0.5.1.1 fansi_0.4.1
[10] magrittr_2.0.1 Rsolnp_1.16 memoise_2.0.0
[13] annotate_1.68.0 modelr_0.1.8 extrafont_0.17
[16] officer_0.3.16 extrafontdb_1.0 colorspace_2.0-0
[19] blob_1.2.1 rvest_0.3.6 haven_2.3.1
[22] xfun_0.20 crayon_1.3.4 RCurl_1.98-1.2
[25] jsonlite_1.7.2 genefilter_1.72.0 survival_3.2-7
[28] glue_1.4.2 gtable_0.3.0 zlibbioc_1.36.0
[31] XVector_0.30.0 webshot_0.5.2 DelayedArray_0.16.0
[34] proj4_1.0-10.1 Rttf2pt1_1.3.8 maps_3.3.0
[37] DBI_1.1.1 Rcpp_1.0.5 viridisLite_0.3.0
[40] xtable_1.8-4 bit_4.0.4 truncnorm_1.0-8
[43] httr_1.4.2 calibrate_1.7.7 ellipsis_0.3.1
[46] mice_3.12.0 farver_2.0.3 pkgconfig_2.0.3
[49] XML_3.99-0.5 dbplyr_2.0.0 locfit_1.5-9.4
[52] labeling_0.4.2 tidyselect_1.1.0 rlang_0.4.10
[55] later_1.1.0.1 AnnotationDbi_1.52.0 munsell_0.5.0
[58] cellranger_1.1.0 tools_4.0.3 cachem_1.0.1
[61] cli_2.2.0 generics_0.1.0 RSQLite_2.2.2
[64] broom_0.7.3 evaluate_0.14 fastmap_1.1.0
[67] yaml_2.2.1 bit64_4.0.5 fs_1.5.0
[70] zip_2.1.1 whisker_0.4 ash_1.0-15
[73] ggrastr_0.2.1 xml2_1.3.2 compiler_4.0.3
[76] rstudioapi_0.13 beeswarm_0.2.3 reprex_1.0.0
[79] geneplotter_1.68.0 stringi_1.5.3 HardyWeinberg_1.7.1
[82] highr_0.8 ps_1.5.0 ggalt_0.4.0
[85] gdtools_0.2.3 lattice_0.20-41 Matrix_1.2-18
[88] vctrs_0.3.6 pillar_1.4.7 lifecycle_0.2.0
[91] data.table_1.13.6 flextable_0.6.2 bitops_1.0-6
[94] httpuv_1.5.5 R6_2.5.0 promises_1.1.1
[97] KernSmooth_2.23-18 vipor_0.4.5 writexl_1.3.1
[100] codetools_0.2-18 MASS_7.3-53 assertthat_0.2.1
[103] chron_2.3-56 rprojroot_2.0.2 withr_2.4.1
[106] GenomeInfoDbData_1.2.4 hms_1.0.0 grid_4.0.3
[109] rmarkdown_2.6 git2r_0.28.0 base64enc_0.1-3
[112] lubridate_1.7.9.2 ggbeeswarm_0.6.0