This package provides a inferential analysis method for detecting differentially expressed CpG sites in MeDIP-seq data. It uses statistical framework and EM algorithm, to identify differentially expressed CpG sites. The methods on this package are described in the article Methylation-level Inferences and Detection of Differential Methylation with Medip-seq Data by Yan Zhou, Jiadi Zhu, Mingtao Zhao, Baoxue Zhang, Chunfu Jiang and Xiyan Yang (2018, pending publication).
SIMD method is developed for jointly analyzing MeDIP-seq and MRE-seq data, which are derived from methylated DNA immunoprecipitation (MeDIP) experiments followed by sequencing (MeDIP-seq) and methyl-sensitive restriction enzymes experiments for unmethylated CpGs (MRE-seq). We have implemented the SIMD method via a set of R functions with the computational intensive parts written in C. We make a R package named SIMD, which is the abbreviation of Statistical Inferences with MeDIP-seq Data, and give a tutorial for the package. The method consist three steps.
Step 1: Data Pre-processing;
Step 2: Calculating p-values of each CpG site by the EM algorithm;
Step 3: Select Significants.
We use a part of a real dataset to illustrate the usage of the SIMD package. The programs can run under the Linux system and windows 10 system. The R versions should larger than 3.5.0.
Before installing SIMD package, the user must install three other R pack- ages, which can be done using the following commands:
install.packages("BiocManager")
BiocManager::install(c('edgeR', 'statmod','methylMnM'))
library(edgeR)
library(statmod)
library(methylMnM)Next, to install the SIMD package into your R environment, start R and enter:
Then, the SIMD package is ready to load.
In order to reproduce the presented SIMD workflow, the package includes the example data sets, which is a part of a real data sets, including:
all_CpGsite_bin_chr18,
three_mre_cpg,
EM2_H1ESB1_MeDIP_sigleCpG,
EM2_H1ESB2_MeDIP_sigleCpG,
H1ESB1_MRE_sigleCpG,
and H1ESB2_MRE_sigleCpG, which is included in file
example_data.RDdata in the data subdirectory of the SIMD package.
The files contain genomic regions from chromosome 18 only, as covered by short reads obtained from a MeDIP and MRE experiment of human H1ESB1 cells.
All output files are in a .bed format. The input file contain the following columns.
For MRE-seq data, we need “+” and “-” strand information in mapping process, which is general located at the sixth column of the input file. Each row represents a mapped read. These information can be extracted from the output file(s) of common mapping tools.
The SIMD program requires users to provide the genome-wide MeDIP-seq and MRE-seq reads and the reference genome. The pre-processing step involves calculation of MeDIP-seq and MRE-seq count and CpG and MRE-specific count in each CpG site. The mainly steps please refer the data pre-processing steps of R package MethylMnM.
In this manual, we use example data located in the data subdirectory of the SIMD package, and the example data is processed by MethylMnM package in advance. The path is:
The CpG count, MRE-CpG count, MeDIP-seq count and MRE-seq count of each site are stored at:
Then we compute the number of actual short reads in each CpG site by the function of EMalgorithm(), which use EM algorithm to infer the actual reads by the observation fragments. The results are saved in file writefile and reportfile. We give an example as follows, the data is from data subdirectory of the SIMD package:
data(example_data)
allcpgfile <- EM_H1ESB1_MeDIP_sigleCpG
readshort <- paste(filepath, "/H1ESB1_MeDIP_18.extended.txt", sep="")
writefile <- paste(dirwrite, "EM2_H1ESB1_MeDIP_sigleCpG.bed", sep="")
reportfile <- paste(dirwrite, "EM2_H1ESB1_MeDIP_sigleCpG_report.bed", sep="")
EMalgorithm(cpgsitefile=readshort, allcpgfile=allcpgfile, category="1",
writefile=writefile, reportfile=reportfile)## [1] 1## V1 V2 V3 V4
## 1 chr18 10692 10694 2.699262074
## 2 chr18 10703 10705 3.464033797
## 3 chr18 10718 10720 5.013880075
## 4 chr18 10730 10732 4.575160390
## 5 chr18 10753 10755 4.367140836
## 6 chr18 10767 10769 3.949686891
## 7 chr18 10787 10789 3.006196402
## 8 chr18 10791 10793 2.721496577
## 9 chr18 10795 10797 2.703602326
## 10 chr18 10815 10817 2.514884741
## 11 chr18 10823 10825 2.335973283
## 12 chr18 10829 10831 2.380368655
## 13 chr18 10831 10833 2.380368655
## 14 chr18 10839 10841 2.226858406
## 15 chr18 10861 10863 1.500303502
## 16 chr18 10869 10871 1.248835637
## 17 chr18 10875 10877 1.136110056
## 18 chr18 10887 10889 0.831184543
## 19 chr18 10921 10923 0.232070026
## 20 chr18 10929 10931 0.232070026
## 21 chr18 10934 10936 0.232070026
## 22 chr18 10937 10939 0.232070026
## 23 chr18 10940 10942 0.179499689
## 24 chr18 10946 10948 0.131977440
## 25 chr18 10948 10950 0.090078076
## 26 chr18 10956 10958 0.090078076
## 27 chr18 10958 10960 0.090078076
## 28 chr18 10963 10965 0.090078076
## 29 chr18 10966 10968 0.056609652
## 30 chr18 10969 10971 0.056609652
## 31 chr18 10975 10977 0.028689994
## 32 chr18 10977 10979 0.028689994
## 33 chr18 10985 10987 0.028689994
## 34 chr18 10987 10989 0.028689994
## 35 chr18 10992 10994 0.028689994
## 36 chr18 10995 10997 0.028689994
## 37 chr18 10998 11000 0.028689994
## 38 chr18 11004 11006 0.007633588
## 39 chr18 11006 11008 0.007633588
## 40 chr18 11014 11016 0.007633588
## 41 chr18 11016 11018 0.007633588
## 42 chr18 11021 11023 0.000000000
## 43 chr18 11024 11026 0.000000000
## 44 chr18 11027 11029 0.000000000
## 45 chr18 11033 11035 0.000000000
## 46 chr18 11035 11037 0.000000000
## 47 chr18 11043 11045 0.000000000
## 48 chr18 11045 11047 0.000000000
## 49 chr18 11050 11052 0.000000000
## 50 chr18 11053 11055 0.000000000
## 51 chr18 11056 11058 0.000000000
## 52 chr18 11062 11064 0.000000000
## 53 chr18 11064 11066 0.000000000
## 54 chr18 11072 11074 0.000000000
## 55 chr18 11074 11076 0.000000000
## 56 chr18 11079 11081 0.000000000
## 57 chr18 11082 11084 0.000000000
## 58 chr18 11085 11087 0.000000000
## 59 chr18 11091 11093 0.000000000
## 60 chr18 11097 11099 0.000000000
## 61 chr18 11099 11101 0.000000000
## 62 chr18 11107 11109 0.000000000
## 63 chr18 11109 11111 0.000000000
## 64 chr18 11114 11116 0.000000000
## 65 chr18 11117 11119 0.000000000
## 66 chr18 11120 11122 0.000000000
## 67 chr18 11126 11128 0.012987013
## 68 chr18 11132 11134 0.052289815
## 69 chr18 11134 11136 0.052289815
## 70 chr18 11142 11144 0.215690742
## 71 chr18 11144 11146 0.215690742
## 72 chr18 11148 11150 0.215690742
## 73 chr18 11154 11156 0.215690742
## 74 chr18 11167 11169 0.752458799
## 75 chr18 11218 11220 1.778161361
## 76 chr18 11230 11232 2.067471968
## 77 chr18 11251 11253 2.381065646
## 78 chr18 11256 11258 2.381065646
## 79 chr18 11323 11325 1.586947232
## 80 chr18 11325 11327 1.586947232
## 81 chr18 11344 11346 1.304016697
## 82 chr18 11354 11356 1.304016697
## 83 chr18 11358 11360 1.080206567
## 84 chr18 11386 11388 1.357217972
## 85 chr18 11400 11402 1.498522320
## 86 chr18 11405 11407 1.498522320
## 87 chr18 11431 11433 1.934716599
## 88 chr18 11511 11513 1.633333333
## 89 chr18 11532 11534 1.250000000
## 90 chr18 11611 11613 1.254824561
## 91 chr18 11667 11669 0.583859649
## 92 chr18 11708 11710 0.583859649
## 93 chr18 11723 11725 1.006224256
## 94 chr18 11739 11741 0.753592677
## 95 chr18 11753 11755 0.427993898
## 96 chr18 11755 11757 0.306372276
## 97 chr18 11761 11763 0.306372276
## 98 chr18 11783 11785 0.306372276
## 99 chr18 11791 11793 0.306372276
## 100 chr18 11793 11795 0.306372276
## 101 chr18 11805 11807 0.165097821
## 102 chr18 11823 11825 0.165097821
## 103 chr18 11825 11827 0.165097821
## 104 chr18 11831 11833 0.240156060
## 105 chr18 11838 11840 0.330145314
## 106 chr18 11849 11851 0.643594231
## 107 chr18 11852 11854 0.643594231
## 108 chr18 11854 11856 0.643594231
## 109 chr18 11863 11865 0.544895607
## 110 chr18 11888 11890 0.498807414
## 111 chr18 11891 11893 0.498807414
## 112 chr18 11893 11895 0.498807414
## 113 chr18 11911 11913 0.465345380
## 114 chr18 11914 11916 0.465345380
## 115 chr18 11939 11941 1.006700023
## 116 chr18 11947 11949 1.006700023
## 117 chr18 11955 11957 1.006700023
## 118 chr18 11962 11964 1.006700023
## 119 chr18 11988 11990 1.328539266
## 120 chr18 11990 11992 1.328539266
## 121 chr18 11996 11998 1.297397153
## 122 chr18 12009 12011 1.260655285
## 123 chr18 12011 12013 1.096611451
## 124 chr18 12022 12024 0.938165718
## 125 chr18 12033 12035 1.079141471
## 126 chr18 12049 12051 1.069957085
## 127 chr18 12052 12054 1.069957085
## 128 chr18 12058 12060 1.069957085
## 129 chr18 12064 12066 1.232874600
## 130 chr18 12067 12069 0.960711886
## 131 chr18 12077 12079 1.014600378
## 132 chr18 12087 12089 0.894328774
## 133 chr18 12101 12103 0.894328774
## 134 chr18 12116 12118 0.799992328
## 135 chr18 12122 12124 0.690645692
## 136 chr18 12139 12141 0.499492449
## 137 chr18 12142 12144 0.499492449
## 138 chr18 12153 12155 0.332982687
## 139 chr18 12160 12162 0.667655848
## 140 chr18 12179 12181 0.635949061
## 141 chr18 12181 12183 0.635949061
## 142 chr18 12204 12206 0.714002799
## 143 chr18 12210 12212 0.768703286
## 144 chr18 12216 12218 0.768703286
## 145 chr18 12223 12225 0.520160207
## 146 chr18 12249 12251 1.649798014
## 147 chr18 12257 12259 1.569077544
## 148 chr18 12272 12274 1.841020424
## 149 chr18 12278 12280 2.145278803
## 150 chr18 12280 12282 2.145278803
## 151 chr18 12293 12295 2.684583553
## 152 chr18 12297 12299 2.881336107
## 153 chr18 12312 12314 2.708051515
## 154 chr18 12336 12338 3.231561757
## 155 chr18 12347 12349 3.557225147
## 156 chr18 12350 12352 3.843967217
## 157 chr18 12361 12363 4.021983139
## 158 chr18 12370 12372 4.738570382
## 159 chr18 12397 12399 4.053435423
## 160 chr18 12436 12438 4.464875891
## 161 chr18 12485 12487 4.155906381
## 162 chr18 12506 12508 4.485778804
## 163 chr18 12527 12529 5.386038747
## 164 chr18 12554 12556 4.969034214
## 165 chr18 12560 12562 4.603399740
## 166 chr18 12570 12572 4.417559580
## 167 chr18 12584 12586 5.875979779
## 168 chr18 12633 12635 6.706979747
## 169 chr18 12709 12711 4.963922243
## 170 chr18 12779 12781 1.448412698
## 171 chr18 12794 12796 0.535714286
## 172 chr18 12855 12857 0.581818182
## 173 chr18 12904 12906 2.254761905
## 174 chr18 12907 12909 2.726406926
## 175 chr18 12953 12955 6.237132651
## 176 chr18 12968 12970 5.809636434
## 177 chr18 13092 13094 3.305587818
## 178 chr18 13118 13120 2.056507937
## 179 chr18 13232 13234 3.563581795
## 180 chr18 13245 13247 3.370989202
## 181 chr18 13262 13264 7.480023511
## 182 chr18 13334 13336 5.346886973
## 183 chr18 13412 13414 4.600000000
## 184 chr18 13548 13550 0.000000000
## 185 chr18 13582 13584 0.000000000
## 186 chr18 13703 13705 0.000000000
## 187 chr18 13866 13868 7.000000000
## 188 chr18 13993 13995 7.500000000
## 189 chr18 14004 14006 7.500000000
## 190 chr18 14235 14237 0.000000000
## 191 chr18 14438 14440 0.000000000
## 192 chr18 14502 14504 0.000000000
## 193 chr18 14522 14524 0.000000000
## 194 chr18 14553 14555 0.000000000
## 195 chr18 14555 14557 0.000000000
## 196 chr18 14576 14578 0.000000000
## 197 chr18 14745 14747 0.000000000
## 198 chr18 14813 14815 0.000000000
## 199 chr18 14839 14841 0.000000000
## 200 chr18 14891 14893 0.000000000
## 201 chr18 15470 15472 0.000000000
## 202 chr18 16066 16068 0.000000000
## 203 chr18 16587 16589 0.000000000
## 204 chr18 17144 17146 0.000000000
## 205 chr18 17914 17916 0.000000000
## 206 chr18 17999 18001 0.000000000
## 207 chr18 18097 18099 0.000000000
## 208 chr18 18113 18115 0.000000000
## 209 chr18 18396 18398 0.000000000
## 210 chr18 18465 18467 0.000000000
## 211 chr18 18543 18545 0.000000000
## 212 chr18 19188 19190 0.000000000
## 213 chr18 19414 19416 0.000000000
## 214 chr18 19944 19946 0.000000000
## 215 chr18 20001 20003 0.000000000
## 216 chr18 20063 20065 0.000000000
## 217 chr18 20095 20097 0.000000000
## 218 chr18 20179 20181 0.000000000
## 219 chr18 20182 20184 0.000000000
## 220 chr18 20685 20687 1.000000000
## 221 chr18 20872 20874 0.000000000
## 222 chr18 21235 21237 0.000000000
## 223 chr18 21329 21331 0.000000000
## 224 chr18 21659 21661 0.000000000
## 225 chr18 21683 21685 0.000000000
## 226 chr18 21705 21707 0.000000000
## 227 chr18 21739 21741 0.000000000
## 228 chr18 21769 21771 0.000000000
## 229 chr18 21778 21780 0.000000000
## 230 chr18 21861 21863 0.000000000
## 231 chr18 21988 21990 0.000000000
## 232 chr18 22336 22338 0.000000000
## 233 chr18 22841 22843 0.000000000
## 234 chr18 23029 23031 0.000000000
## 235 chr18 23066 23068 0.000000000
## 236 chr18 23281 23283 0.000000000
## 237 chr18 23401 23403 0.000000000
## 238 chr18 23439 23441 0.000000000
## 239 chr18 23456 23458 0.000000000
## 240 chr18 23476 23478 0.000000000
## 241 chr18 23515 23517 0.000000000
## 242 chr18 23557 23559 0.000000000
## 243 chr18 23851 23853 0.000000000
## 244 chr18 23927 23929 0.000000000
## 245 chr18 24575 24577 0.000000000
## 246 chr18 25082 25084 0.000000000
## 247 chr18 25270 25272 0.000000000
## 248 chr18 25286 25288 0.000000000
## 249 chr18 25421 25423 0.000000000
## 250 chr18 25660 25662 0.000000000
## 251 chr18 25865 25867 0.000000000
## 252 chr18 25871 25873 0.000000000
## 253 chr18 26060 26062 0.000000000
## 254 chr18 26187 26189 0.000000000
## 255 chr18 26196 26198 0.000000000
## 256 chr18 26327 26329 0.000000000
## 257 chr18 26627 26629 0.200000000
## 258 chr18 26661 26663 0.733333333
## 259 chr18 26690 26692 0.733333333
## 260 chr18 26713 26715 0.166666667
## 261 chr18 26792 26794 0.166666667
## 262 chr18 26802 26804 0.000000000
## 263 chr18 26807 26809 0.000000000
## 264 chr18 26845 26847 0.000000000
## 265 chr18 26847 26849 0.000000000
## 266 chr18 26858 26860 0.000000000
## 267 chr18 26944 26946 0.000000000
## 268 chr18 26988 26990 0.000000000
## 269 chr18 27167 27169 0.000000000
## 270 chr18 27208 27210 0.000000000
## 271 chr18 27819 27821 0.344720497
## 272 chr18 27828 27830 0.344720497
## 273 chr18 27847 27849 0.919772257
## 274 chr18 27915 27917 1.343726708
## 275 chr18 27929 27931 1.343726708
## 276 chr18 27947 27949 0.902380952
## 277 chr18 27970 27972 0.327142857
## 278 chr18 27982 27984 0.327142857
## 279 chr18 27992 27994 0.146666667
## 280 chr18 28007 28009 0.000000000
## 281 chr18 28022 28024 0.000000000
## 282 chr18 28032 28034 0.000000000
## 283 chr18 28040 28042 0.000000000
## 284 chr18 28053 28055 0.000000000
## 285 chr18 28142 28144 0.000000000
## 286 chr18 28151 28153 0.000000000
## 287 chr18 28171 28173 0.000000000
## 288 chr18 28204 28206 0.000000000
## 289 chr18 28221 28223 0.000000000
## 290 chr18 28234 28236 0.000000000
## 291 chr18 28368 28370 0.000000000
## 292 chr18 28379 28381 0.000000000
## 293 chr18 28387 28389 0.000000000
## 294 chr18 28426 28428 0.000000000
## 295 chr18 28495 28497 0.000000000
## 296 chr18 28501 28503 0.000000000
## 297 chr18 28524 28526 0.000000000
## 298 chr18 28544 28546 0.000000000
## 299 chr18 28562 28564 0.000000000
## 300 chr18 28666 28668 0.000000000
## 301 chr18 28733 28735 0.000000000
## 302 chr18 28833 28835 0.000000000
## 303 chr18 28853 28855 0.000000000
## 304 chr18 28895 28897 0.000000000
## 305 chr18 28912 28914 0.000000000
## 306 chr18 29146 29148 0.000000000
## 307 chr18 29221 29223 0.000000000
## 308 chr18 29422 29424 0.000000000
## 309 chr18 29431 29433 0.000000000
## 310 chr18 29652 29654 0.000000000
## 311 chr18 29918 29920 0.000000000
## 312 chr18 29976 29978 0.000000000
## 313 chr18 30288 30290 0.000000000
## 314 chr18 30385 30387 0.000000000
## 315 chr18 30591 30593 0.000000000
## 316 chr18 30870 30872 0.000000000
## 317 chr18 30893 30895 0.000000000
## 318 chr18 30936 30938 0.000000000
## 319 chr18 31098 31100 0.000000000
## 320 chr18 31461 31463 0.000000000
## 321 chr18 31914 31916 0.000000000
## 322 chr18 31930 31932 0.000000000
## 323 chr18 32210 32212 0.000000000
## 324 chr18 32477 32479 0.000000000
## 325 chr18 32732 32734 0.000000000
## 326 chr18 32824 32826 0.000000000
## 327 chr18 33717 33719 0.000000000
## 328 chr18 34235 34237 0.000000000
## 329 chr18 34281 34283 0.000000000
## 330 chr18 34319 34321 0.000000000
## 331 chr18 34334 34336 0.000000000
## 332 chr18 34857 34859 0.000000000
## 333 chr18 34972 34974 0.000000000
## 334 chr18 34976 34978 0.000000000
## 335 chr18 35523 35525 0.000000000
## 336 chr18 35845 35847 0.000000000
## 337 chr18 35851 35853 0.000000000
## 338 chr18 35863 35865 0.000000000
## 339 chr18 35929 35931 0.000000000
## 340 chr18 35947 35949 0.000000000
## 341 chr18 35952 35954 0.000000000
## 342 chr18 35963 35965 0.000000000
## 343 chr18 35990 35992 0.000000000
## 344 chr18 35997 35999 0.000000000
## 345 chr18 36062 36064 0.206349206
## 346 chr18 36077 36079 0.206349206
## 347 chr18 36097 36099 0.206349206
## 348 chr18 36111 36113 0.732698413
## 349 chr18 36114 36116 0.732698413
## 350 chr18 36153 36155 0.732698413
## 351 chr18 36157 36159 0.382857143
## 352 chr18 36171 36173 0.160000000
## 353 chr18 36178 36180 0.160000000
## 354 chr18 36190 36192 0.160000000
## 355 chr18 36218 36220 0.160000000
## 356 chr18 36239 36241 0.160000000
## 357 chr18 36285 36287 0.000000000
## 358 chr18 36425 36427 0.000000000
## 359 chr18 36557 36559 0.000000000
## 360 chr18 36660 36662 0.000000000
## 361 chr18 36697 36699 0.000000000
## 362 chr18 36743 36745 0.000000000
## 363 chr18 36840 36842 0.000000000
## 364 chr18 36861 36863 0.000000000
## 365 chr18 36987 36989 0.000000000
## 366 chr18 36996 36998 0.000000000
## 367 chr18 37023 37025 0.000000000
## 368 chr18 37028 37030 0.000000000
## 369 chr18 37054 37056 0.000000000
## 370 chr18 37083 37085 0.000000000
## 371 chr18 37170 37172 0.000000000
## 372 chr18 37321 37323 0.000000000
## 373 chr18 37410 37412 0.000000000
## 374 chr18 37677 37679 0.000000000
## 375 chr18 37720 37722 0.000000000
## 376 chr18 37749 37751 0.000000000
## 377 chr18 37915 37917 0.000000000
## 378 chr18 38642 38644 0.000000000
## 379 chr18 38982 38984 0.000000000
## 380 chr18 39033 39035 0.000000000
## 381 chr18 39210 39212 0.000000000
## 382 chr18 39521 39523 0.000000000
## 383 chr18 39801 39803 0.000000000
## 384 chr18 39887 39889 0.000000000
## 385 chr18 40212 40214 0.000000000
## 386 chr18 40339 40341 0.000000000
## 387 chr18 40502 40504 0.000000000
## 388 chr18 40680 40682 0.000000000
## 389 chr18 41251 41253 0.000000000
## 390 chr18 41359 41361 0.000000000
## 391 chr18 41372 41374 0.000000000
## 392 chr18 41407 41409 0.000000000
## 393 chr18 41552 41554 0.000000000
## 394 chr18 41575 41577 0.000000000
## 395 chr18 41740 41742 0.000000000
## 396 chr18 41953 41955 0.000000000
## 397 chr18 42652 42654 0.000000000
## 398 chr18 42831 42833 0.000000000
## 399 chr18 43156 43158 0.000000000
## 400 chr18 43562 43564 0.000000000
## 401 chr18 43610 43612 0.000000000
## 402 chr18 43619 43621 0.000000000
## 403 chr18 43640 43642 0.000000000
## 404 chr18 43704 43706 1.000000000
## 405 chr18 44032 44034 0.000000000
## 406 chr18 44211 44213 0.000000000
## 407 chr18 44548 44550 0.000000000
## 408 chr18 44589 44591 0.000000000
## 409 chr18 44611 44613 0.000000000
## 410 chr18 44795 44797 0.000000000
## 411 chr18 44798 44800 0.000000000
## 412 chr18 44811 44813 0.000000000
## 413 chr18 45139 45141 0.000000000
## 414 chr18 45322 45324 0.000000000
## 415 chr18 45425 45427 0.000000000
## 416 chr18 45676 45678 1.000000000
## 417 chr18 45678 45680 1.000000000
## 418 chr18 45750 45752 0.000000000
## 419 chr18 45867 45869 0.000000000
## 420 chr18 45950 45952 0.000000000
## 421 chr18 46049 46051 0.333333333
## 422 chr18 46140 46142 0.333333333
## 423 chr18 46191 46193 0.333333333
## 424 chr18 46232 46234 0.500000000
## 425 chr18 46240 46242 0.500000000
## 426 chr18 46627 46629 1.666666667
## 427 chr18 46655 46657 0.333333333
## 428 chr18 46797 46799 0.000000000
## 429 chr18 46928 46930 0.000000000
## 430 chr18 47085 47087 1.000000000
## 431 chr18 47147 47149 0.000000000
## 432 chr18 47167 47169 0.000000000
## 433 chr18 47253 47255 0.000000000
## 434 chr18 47278 47280 0.000000000
## 435 chr18 47312 47314 0.000000000
## 436 chr18 47406 47408 0.000000000
## 437 chr18 47433 47435 0.000000000
## 438 chr18 47437 47439 0.000000000
## 439 chr18 47475 47477 0.000000000
## 440 chr18 47490 47492 0.000000000
## 441 chr18 47496 47498 0.000000000
## 442 chr18 47523 47525 0.200000000
## 443 chr18 47527 47529 0.200000000
## 444 chr18 47552 47554 0.200000000
## 445 chr18 47608 47610 0.200000000
## 446 chr18 47648 47650 0.200000000
## 447 chr18 47667 47669 0.142857143
## 448 chr18 47679 47681 0.142857143
## 449 chr18 47685 47687 0.142857143
## 450 chr18 47765 47767 0.142857143
## 451 chr18 47788 47790 0.142857143
## 452 chr18 47802 47804 0.142857143
## 453 chr18 47807 47809 0.142857143
## 454 chr18 47879 47881 0.000000000
## 455 chr18 47897 47899 0.000000000
## 456 chr18 47919 47921 0.000000000
## 457 chr18 47939 47941 0.000000000
## 458 chr18 47947 47949 0.000000000
## 459 chr18 47961 47963 0.000000000
## 460 chr18 47972 47974 0.000000000
## 461 chr18 48002 48004 0.000000000
## 462 chr18 48106 48108 0.250000000
## 463 chr18 48111 48113 0.250000000
## 464 chr18 48136 48138 0.250000000
## 465 chr18 48138 48140 0.250000000
## 466 chr18 48171 48173 0.250000000
## 467 chr18 48189 48191 0.250000000
## 468 chr18 48196 48198 0.250000000
## 469 chr18 48208 48210 0.250000000
## 470 chr18 48257 48259 0.000000000
## 471 chr18 48369 48371 0.000000000
## 472 chr18 48385 48387 0.000000000
## 473 chr18 48394 48396 0.000000000
## 474 chr18 48396 48398 0.000000000
## 475 chr18 48410 48412 0.000000000
## 476 chr18 48432 48434 0.000000000
## 477 chr18 48464 48466 1.000000000
## 478 chr18 48472 48474 1.000000000
## 479 chr18 48475 48477 1.000000000
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## 1059 chr18 84721 84723 2.039845261
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## 1062 chr18 84786 84788 1.473838138
## 1063 chr18 84805 84807 2.018067905
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## 1069 chr18 84987 84989 0.498725074
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## 1072 chr18 85047 85049 3.433993952
## 1073 chr18 85094 85096 3.643517762
## 1074 chr18 85187 85189 4.629270186
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## 1165 chr18 90897 90899 1.141025641
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## 1588 chr18 119956 119958 0.363636364
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## 1628 chr18 125245 125247 0.153846154
## 1629 chr18 125247 125249 0.153846154
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## 1632 chr18 125369 125371 0.656177156
## 1633 chr18 125379 125381 0.656177156
## 1634 chr18 125383 125385 0.656177156
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## 1914 chr18 149255 149257 1.402597403
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## 1917 chr18 149390 149392 2.282284382
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## 1953 chr18 153209 153211 1.000000000
## 1954 chr18 153295 153297 0.000000000
## 1955 chr18 153352 153354 0.000000000
## 1956 chr18 153482 153484 0.000000000
## 1957 chr18 153521 153523 0.140740741
## 1958 chr18 153545 153547 0.304861111
## 1959 chr18 153550 153552 0.304861111
## 1960 chr18 153582 153584 0.304861111
## 1961 chr18 153613 153615 0.580394525
## 1962 chr18 153636 153638 0.580394525
## 1963 chr18 153640 153642 0.580394525
## 1964 chr18 153645 153647 0.580394525
## 1965 chr18 153661 153663 0.324184783
## 1966 chr18 153683 153685 0.149456522
## 1967 chr18 153689 153691 0.149456522
## 1968 chr18 153791 153793 0.062500000
## 1969 chr18 153875 153877 1.247282609
## 1970 chr18 153887 153889 1.247282609
## 1971 chr18 153891 153893 1.247282609
## 1972 chr18 153951 153953 1.559782609
## 1973 chr18 153979 153981 0.635869565
## 1974 chr18 154304 154306 0.000000000
## 1975 chr18 154357 154359 1.300000000
## 1976 chr18 154365 154367 1.300000000
## 1977 chr18 154484 154486 0.981818182
## 1978 chr18 154522 154524 0.792344498
## 1979 chr18 154527 154529 0.792344498
## 1980 chr18 154536 154538 0.792344498
## 1981 chr18 154554 154556 0.792344498
## 1982 chr18 154616 154618 0.196172249
## 1983 chr18 154626 154628 0.052631579
## 1984 chr18 154670 154672 0.500000000
## 1985 chr18 154687 154689 0.500000000
## 1986 chr18 154882 154884 1.000000000
## 1987 chr18 155278 155280 0.444444444
## 1988 chr18 155287 155289 1.000000000
## 1989 chr18 155308 155310 2.000000000
## 1990 chr18 155413 155415 0.555555556
## 1991 chr18 155565 155567 0.000000000
## 1992 chr18 155576 155578 0.000000000
## 1993 chr18 155660 155662 0.000000000
## 1994 chr18 155676 155678 0.000000000
## 1995 chr18 155704 155706 0.000000000
## 1996 chr18 155797 155799 0.000000000
## 1997 chr18 155847 155849 0.000000000
## 1998 chr18 155868 155870 0.000000000
## 1999 chr18 155915 155917 0.000000000
## 2000 chr18 155969 155971 0.000000000In order to detect different methylated CpG sites, we should calculate p-value of each site. The below codes are calculate p-value of each site with function EMtest(). The input files which include “datafile”, “cpgfile” and “mrecpgfile” are should have been generated by Step 1. The output file “writefile” will owneleven columns, that is, “chr”, “chrSt”,“chrEnd”, “Medip1”,“Medip2”, “MRE1”, “MRE2”, “cg”,“mrecg”,“pvalue”,“Ts”. We also output a reportfile which will include parameters “s1/s2”; “s3/s4”; “N1”; “N2”; “N3”; “N4”; “c1”; “c2” and “Spend time”.
data(example_data)
data1 <- EM2_H1ESB1_MeDIP_sigleCpG
data2 <- EM2_H1ESB2_MeDIP_sigleCpG
data3 <- H1ESB1_MRE_sigleCpG
data4 <- H1ESB2_MRE_sigleCpG
datafile <- cbind(data1,data2,data3,data4)
allcpg <- all_CpGsite_bin_chr18
mrecpg <- three_mre_cpg
dirwrite <-paste(setwd(getwd()), "/", sep="")
writefile <- paste(dirwrite, "pval_EM_H1ESB1_H1ESB21.bed", sep="")
reportfile <- paste(dirwrite, "report_pvalH1ESB1_H1ESB21.bed", sep="")
EMtest(datafile=datafile, chrstring=NULL, cpgfile=allcpg,mrecpgfile=mrecpg,
writefile=writefile, reportfile=reportfile,mreratio=3/7, psd=2,
mkadded=1, f=1)## reading data ....## adjustment weight f is :## [1] 1## Top line of dataset:## V1 V2 V3 V4 data2[, 4] data3[, 4] data4[, 4]
## 1 chr18 10692 10694 8.824992 7.185892 0 0
## V1 V2 V3 V4
## 1 chr18 10692 10694 1
## V1 V2 V3 V4
## 1 chr18 10692 10694 0## Computing p-values for only MeDIP-seq windows: 1286## Computing p-values for MeDIP-seq
## and MRE-seq windows: 714## Exact compute p-values with product binomial distribution.## Output to writefile.## The End.After getting p-values, we choose differentially expressed methylated CpG sites with p-values. The input file is the file which have been generated by Step 2. Then we assume the sites to be differentially expressed methylated CpGs when p-values less than pre-setting cutoffs, such as 10−3 or 10−5.