Last updated: 2023-09-04

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Knit directory: RA_Tcell_omics/analysis/

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Load libraries

Global variables

DMR analysis to identify significant DMRs that contain specific regions associated with genes (use hg19 annotation)

Preprocessing

Load data

Get unique symbol

Get interested gene list

Process methylation dataset

[1] 20884    23

Differential methylation analysis on probe level

SVA to identify unknown confounder

Number of significant surrogate variables is:  3 
Iteration (out of 5 ):1  2  3  4  5

DE test using limma

Using dmrff to identify DMRs

[dmrff.candidates] Mon Sep  4 15:16:22 2023 Found  1087  candidate regions.

Annotate MDRs

Save significant DMRs with annotation

    symbol  DMR    site   chr     start       end number_CpG  estimate
1 SLC16A11 dmr1    Body chr17   6945510   6946086          4 0.9815272
2   CACNG2 dmr2  TSS200 chr22  37099095  37099785          7 0.6132989
3      GDA dmr3   5'UTR  chr9  74764261  74764263          2 1.0916481
4      RGN dmr4 TSS1500  chrX  46937571  46938148          7 0.9524670
5     NOS1 dmr5 1stExon chr12 117799370 117799749          3 0.6260553
6   CYP1B1 dmr6    Body  chr2  38300537  38300885          4 1.0469769
       p.value     p.adjust
1 8.640059e-15 1.981943e-10
2 1.004762e-07 2.304823e-03
3 1.316342e-07 3.019557e-03
4 2.047986e-07 4.697875e-03
5 2.320634e-07 5.323303e-03
6 1.351948e-06 3.101233e-02

Download xlsx table: DMR_regions.xlsx

Visuaize enhancer/promoter methylation status per gene

Download plots in zip file: plot_DMR.zip

DMR analysis to identify significant DMRs that contain GeneHancer regions

Process enhancer list

Create genomic ranges object

Find GC probs in the enhancer region

Perform differential analysis

Process methylation dataset

[1] 85102    23

SVA to identify unknwon confounder

Number of significant surrogate variables is:  3 
Iteration (out of 5 ):1  2  3  4  5

DE test using limma

Add mean difference of beta values

Get enhancer/promoter regions that contains differentially expressed probes

Comine the two tables

Using dmrff to identify DMRs

[dmrff.candidates] Mon Sep  4 15:17:16 2023 Found  4660  candidate regions.

Annotate DMRs

        gene  enhancerId           feature   chr    start      end number_CpG
1   SLC16A11 GH17J007041 Promoter/Enhancer chr17  6945510  6946086          4
1.1 SLC16A13 GH17J007041 Promoter/Enhancer chr17  6945510  6946086          4
2        DDC GH07J050790 Promoter/Enhancer  chr7 50861467 50861750         11
3       USP8 GH15J050181 Promoter/Enhancer chr15 50473854 50474221          6
3.1      HDC GH15J050181 Promoter/Enhancer chr15 50473854 50474221          6
4       GART GH21J033400 Promoter/Enhancer chr21 34775001 34775045          5
     estimate      p.value     p.adjust
1   0.9974066 2.710935e-15 2.566333e-10
1.1 0.9974066 2.710935e-15 2.566333e-10
2   0.8361415 5.662111e-13 5.360094e-08
3   0.8601667 1.795356e-11 1.699592e-06
3.1 0.8601667 1.795356e-11 1.699592e-06
4   1.6298080 4.465063e-10 4.226896e-05

Download xlsx table: DMR_GeneHancer.xlsx

Visuaize enhancer/promoter methylation status per gene

Download plots in zip file: plot_geneEnhancer.zip

Heatmap visualization of all significant DMRs

Example plot of enhancers

pdf 
  2 


R version 4.2.0 (2022-04-22)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Big Sur/Monterey 10.16

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] grid      stats4    stats     graphics  grDevices utils     datasets 
[8] methods   base     

other attached packages:
 [1] cowplot_1.1.1               ComplexHeatmap_2.12.1      
 [3] gridExtra_2.3               sva_3.44.0                 
 [5] BiocParallel_1.30.3         genefilter_1.78.0          
 [7] mgcv_1.8-40                 nlme_3.1-158               
 [9] forcats_0.5.1               stringr_1.4.1              
[11] dplyr_1.0.9                 purrr_0.3.4                
[13] readr_2.1.2                 tidyr_1.2.0                
[15] tibble_3.1.8                ggplot2_3.4.1              
[17] tidyverse_1.3.2             pheatmap_1.0.12            
[19] SummarizedExperiment_1.26.1 Biobase_2.56.0             
[21] GenomicRanges_1.48.0        GenomeInfoDb_1.32.2        
[23] IRanges_2.30.0              S4Vectors_0.34.0           
[25] BiocGenerics_0.42.0         MatrixGenerics_1.8.1       
[27] matrixStats_0.62.0          limma_3.52.2               

loaded via a namespace (and not attached):
  [1] utf8_1.2.2             shinydashboard_0.7.2   tidyselect_1.1.2      
  [4] RSQLite_2.2.15         AnnotationDbi_1.58.0   htmlwidgets_1.5.4     
  [7] maxstat_0.7-25         munsell_0.5.0          ragg_1.2.2            
 [10] codetools_0.2-18       DT_0.23                withr_2.5.0           
 [13] colorspace_2.0-3       highr_0.9              knitr_1.39            
 [16] rstudioapi_0.13        ggsignif_0.6.3         labeling_0.4.2        
 [19] git2r_0.30.1           slam_0.1-50            GenomeInfoDbData_1.2.8
 [22] KMsurv_0.1-5           bit64_4.0.5            farver_2.1.1          
 [25] rprojroot_2.0.3        vctrs_0.5.2            generics_0.1.3        
 [28] TH.data_1.1-1          xfun_0.31              sets_1.0-21           
 [31] R6_2.5.1               doParallel_1.0.17      ggbeeswarm_0.6.0      
 [34] clue_0.3-61            locfit_1.5-9.6         bitops_1.0-7          
 [37] cachem_1.0.6           fgsea_1.22.0           DelayedArray_0.22.0   
 [40] assertthat_0.2.1       promises_1.2.0.1       scales_1.2.0          
 [43] multcomp_1.4-19        googlesheets4_1.0.0    beeswarm_0.4.0        
 [46] gtable_0.3.0           Cairo_1.6-0            dmrff_1.1.0           
 [49] sandwich_3.0-2         workflowr_1.7.0        rlang_1.0.6           
 [52] systemfonts_1.0.4      GlobalOptions_0.1.2    splines_4.2.0         
 [55] rstatix_0.7.0          gargle_1.2.0           broom_1.0.0           
 [58] yaml_2.3.5             abind_1.4-5            modelr_0.1.8          
 [61] backports_1.4.1        httpuv_1.6.6           tools_4.2.0           
 [64] relations_0.6-12       ellipsis_0.3.2         gplots_3.1.3          
 [67] jquerylib_0.1.4        RColorBrewer_1.1-3     Rcpp_1.0.9            
 [70] visNetwork_2.1.0       zlibbioc_1.42.0        RCurl_1.98-1.7        
 [73] ggpubr_0.4.0           GetoptLong_1.0.5       zoo_1.8-10            
 [76] haven_2.5.0            cluster_2.1.3          exactRankTests_0.8-35 
 [79] fs_1.5.2               magrittr_2.0.3         magick_2.7.3          
 [82] data.table_1.14.8      circlize_0.4.15        reprex_2.0.1          
 [85] survminer_0.4.9        googledrive_2.0.0      mvtnorm_1.1-3         
 [88] hms_1.1.1              shinyjs_2.1.0          mime_0.12             
 [91] evaluate_0.15          xtable_1.8-4           XML_3.99-0.10         
 [94] readxl_1.4.0           shape_1.4.6            compiler_4.2.0        
 [97] writexl_1.4.0          KernSmooth_2.23-20     crayon_1.5.2          
[100] htmltools_0.5.4        later_1.3.0            tzdb_0.3.0            
[103] lubridate_1.8.0        DBI_1.1.3              dbplyr_2.2.1          
[106] MASS_7.3-58            jyluMisc_0.1.5         Matrix_1.5-4          
[109] car_3.1-0              cli_3.4.1              marray_1.74.0         
[112] parallel_4.2.0         igraph_1.3.4           pkgconfig_2.0.3       
[115] km.ci_0.5-6            piano_2.12.0           xml2_1.3.3            
[118] foreach_1.5.2          annotate_1.74.0        vipor_0.4.5           
[121] bslib_0.4.1            XVector_0.36.0         drc_3.0-1             
[124] rvest_1.0.2            digest_0.6.30          Biostrings_2.64.0     
[127] rmarkdown_2.14         cellranger_1.1.0       fastmatch_1.1-3       
[130] survMisc_0.5.6         edgeR_3.38.1           shiny_1.7.4           
[133] gtools_3.9.3           rjson_0.2.21           lifecycle_1.0.3       
[136] jsonlite_1.8.3         carData_3.0-5          fansi_1.0.3           
[139] pillar_1.8.0           lattice_0.20-45        KEGGREST_1.36.3       
[142] fastmap_1.1.0          httr_1.4.3             plotrix_3.8-2         
[145] survival_3.4-0         glue_1.6.2             png_0.1-7             
[148] iterators_1.0.14       bit_4.0.4              stringi_1.7.8         
[151] sass_0.4.2             blob_1.2.3             textshaping_0.3.6     
[154] caTools_1.18.2         memoise_2.0.1