Dear EPIC users,

I have question about EPIC v2. I analyzed my data using minfi and got beta values for all the probes that passed quality ect. Now I would like to create bigwig files so that I can look at my data in UCSC or another genome browser. However, that is where I encountered a problem.

This is the code I used:

library(IlluminaHumanMethylationEPICv2anno.20a1.hg38)
library(BSgenome.Hsapiens.UCSC.hg38)
library(rtracklayer)
data(Locations)

annotation <- getAnnotation(IlluminaHumanMethylationEPICv2anno.20a1.hg38)
annotation_df <- as.data.frame(annotation)

positions_EPIC=data.frame(row.names = row.names(annotation_df),
                     chr=annotation_df$chr,
                     start=annotation_df$pos,
                     end=ifelse(annotation_df$strand == "+", annotation_df$pos + 1, annotation_df$pos - 1),
                     strand=annotation_df$strand)

# Function to swap start and end if start is greater than end
swap_if_needed <- function(start, end) {
  if (start > end) {
    temp <- start
    start <- end
    end <- temp
  }
  return(c(start, end))
}

# Apply the function to each row of the data frame
for (i in 1:nrow(positions_EPIC)) {
  result <- swap_if_needed(positions_EPIC$start[i], positions_EPIC$end[i])
  positions_EPIC$start[i] <- result[1]
  positions_EPIC$end[i] <- result[2]
}

# Print the updated data frame
print(positions_EPIC)

hg38info=seqinfo(BSgenome.Hsapiens.UCSC.hg38)
mybeta2 = na.omit(mybeta)

for (i in 1:ncol(mybeta2)){
  mySample=merge(positions_EPIC,mybeta2[,i],by="row.names")
  names(mySample)[6]="score"
  mySample$Row.names=c()
  print(mySample)
  name=colnames(mybeta2)[i]
  write.table(mySample[,c("chr","start","end","score")],
              sep="\t",
              row.names = FALSE,
              col.names = FALSE,
              quote = FALSE,
              file=paste(name,".bedGraph",sep=""))
  mySample$strand = NULL
  print(mySample)
  myRanges=makeGRangesFromDataFrame(mySample,
                                    keep.extra.columns = TRUE)
  seqinfo(myRanges)=hg38info
  export.bw(myRanges,paste0(name,".bw"))
}

this worked till the very last line - export.bw

Error in FUN(extractROWS(unlisted_X, IRanges(X_elt_start[i], X_elt_end[i])),  : 
  BigWig ranges cannot overlap
In addition: Warning message:
In valid.GenomicRanges.seqinfo(x, suggest.trim = TRUE) :
  GRanges object contains 4 out-of-bound ranges located on sequences chr6,
  chr11, chr14, and chr18. Note that ranges located on a sequence whose length
  is unknown (NA) or on a circular sequence are not considered out-of-bound
  (use seqlengths() and isCircular() to get the lengths and circularity flags
  of the underlying sequences). You can use trim() to trim these ranges. See
  ?`trim,GenomicRanges-method` for more information.

Then I decided to skip coupe of list lines and just write all the bedgraph files (since that worked), and then I tried to convert them using bedgraphToBogwig finction from UCSC but then error was different - End coordinate 140288269 bigger than chr11 size of 135086622 line 168183 of file

And indeed in EPIC annotation this is the position of the prob/cpg

however that is out of the coordinates in hg38 genome

Does anyone knows why is this, and how to generate bigwig files?

Thank you all, Maja

> sessionInfo( )
R version 4.4.0 (2024-04-24 ucrt)
Platform: x86_64-w64-mingw32/x64
Running under: Windows 10 x64 (build 19045)

Matrix products: default


locale:
[1] LC_COLLATE=Dutch_Netherlands.utf8  LC_CTYPE=Dutch_Netherlands.utf8   
[3] LC_MONETARY=Dutch_Netherlands.utf8 LC_NUMERIC=C                      
[5] LC_TIME=Dutch_Netherlands.utf8    

time zone: Europe/Amsterdam
tzcode source: internal

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

other attached packages:
 [1] DNAmArray_2.0.0                                   
 [2] pls_2.8-3                                         
 [3] FDb.InfiniumMethylation.hg19_2.2.0                
 [4] org.Hs.eg.db_3.19.1                               
 [5] TxDb.Hsapiens.UCSC.hg19.knownGene_3.2.2           
 [6] GenomicFeatures_1.56.0                            
 [7] AnnotationDbi_1.66.0                              
 [8] BiocManager_1.30.23                               
 [9] devtools_2.4.5                                    
[10] usethis_2.2.3                                     
[11] IlluminaHumanMethylationEPICv2manifest_1.0.0      
[12] BSgenome.Hsapiens.UCSC.hg38_1.4.5                 
[13] BSgenome_1.72.0                                   
[14] rtracklayer_1.64.0                                
[15] BiocIO_1.14.0                                     
[16] IlluminaHumanMethylationEPICv2anno.20a1.hg38_1.0.0
[17] minfi_1.50.0                                      
[18] bumphunter_1.46.0                                 
[19] locfit_1.5-9.10                                   
[20] iterators_1.0.14                                  
[21] foreach_1.5.2                                     
[22] Biostrings_2.72.1                                 
[23] XVector_0.44.0                                    
[24] SummarizedExperiment_1.34.0                       
[25] Biobase_2.64.0                                    
[26] MatrixGenerics_1.16.0                             
[27] matrixStats_1.3.0                                 
[28] GenomicRanges_1.56.1                              
[29] GenomeInfoDb_1.40.1                               
[30] IRanges_2.38.0                                    
[31] S4Vectors_0.42.0                                  
[32] BiocGenerics_0.50.0                               
[33] lubridate_1.9.3                                   
[34] forcats_1.0.0                                     
[35] stringr_1.5.1                                     
[36] dplyr_1.1.4                                       
[37] purrr_1.0.2                                       
[38] tidyr_1.3.1                                       
[39] tibble_3.2.1                                      
[40] ggplot2_3.5.1                                     
[41] tidyverse_2.0.0                                   
[42] readr_2.1.5                                       

loaded via a namespace (and not attached):
  [1] splines_4.4.0             later_1.3.2               bitops_1.0-7             
  [4] preprocessCore_1.66.0     XML_3.99-0.16.1           lifecycle_1.0.4          
  [7] processx_3.8.4            lattice_0.22-6            vroom_1.6.5              
 [10] MASS_7.3-60.2             base64_2.0.1              scrime_1.3.5             
 [13] magrittr_2.0.3            limma_3.60.3              yaml_2.3.8               
 [16] remotes_2.5.0             httpuv_1.6.15             doRNG_1.8.6              
 [19] askpass_1.2.0             sessioninfo_1.2.2         pkgbuild_1.4.4           
 [22] DBI_1.2.3                 RColorBrewer_1.1-3        abind_1.4-5              
 [25] pkgload_1.4.0             zlibbioc_1.50.0           quadprog_1.5-8           
 [28] RCurl_1.98-1.14           GenomeInfoDbData_1.2.12   genefilter_1.86.0        
 [31] annotate_1.82.0           DelayedMatrixStats_1.26.0 codetools_0.2-20         
 [34] DelayedArray_0.30.1       xml2_1.3.6                tidyselect_1.2.1         
 [37] UCSC.utils_1.0.0          beanplot_1.3.1            illuminaio_0.46.0        
 [40] GenomicAlignments_1.40.0  jsonlite_1.8.8            multtest_2.60.0          
 [43] ellipsis_0.3.2            survival_3.7-0            tools_4.4.0              
 [46] Rcpp_1.0.12               glue_1.7.0                SparseArray_1.4.8        
 [49] HDF5Array_1.32.0          withr_3.0.0               fastmap_1.2.0            
 [52] rhdf5filters_1.16.0       fansi_1.0.6               openssl_2.2.0            
 [55] callr_3.7.6               digest_0.6.35             timechange_0.3.0         
 [58] R6_2.5.1                  mime_0.12                 colorspace_2.1-0         
 [61] RSQLite_2.3.7             utf8_1.2.4                generics_0.1.3           
 [64] data.table_1.15.4         httr_1.4.7                htmlwidgets_1.6.4        
 [67] S4Arrays_1.4.1            pkgconfig_2.0.3           gtable_0.3.5             
 [70] blob_1.2.4                siggenes_1.78.0           htmltools_0.5.8.1        
 [73] profvis_0.3.8             scales_1.3.0              png_0.1-8                
 [76] rstudioapi_0.16.0         reshape2_1.4.4            tzdb_0.4.0               
 [79] rjson_0.2.21              nlme_3.1-164              curl_5.2.1               
 [82] cachem_1.1.0              rhdf5_2.48.0              miniUI_0.1.1.1           
 [85] restfulr_0.0.15           desc_1.4.3                GEOquery_2.72.0          
 [88] pillar_1.9.0              grid_4.4.0                reshape_0.8.9            
 [91] vctrs_0.6.5               urlchecker_1.0.1          promises_1.3.0           
 [94] xtable_1.8-4              cli_3.6.1                 compiler_4.4.0           
 [97] Rsamtools_2.20.0          rlang_1.1.3               crayon_1.5.3             
[100] rngtools_1.5.2            nor1mix_1.3-3             mclust_6.1.1             
[103] ps_1.7.6                  plyr_1.8.9                fs_1.6.4                 
[106] stringi_1.8.4             BiocParallel_1.38.0       htm2txt_2.2.2            
[109] munsell_0.5.1             Matrix_1.7-0              hms_1.1.3                
[112] sparseMatrixStats_1.16.0  bit64_4.0.5               Rhdf5lib_1.26.0          
[115] KEGGREST_1.44.1           statmod_1.5.0             shiny_1.8.1.1            
[118] memoise_2.0.1             bit_4.0.5