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Using Methylation Tracks

  • Version 1* is when all contexts are in a single BigWig file
    • This caused problems with zooming in the browser, so version 2 was developed as a fix.
    • Version 1 is still supported, but not recommended for use.
  • Version 2 uses three BigWig files, one for each methylation context.
    • The files need the same base name, like my-file.bw, and contexts are specified as additional extensions, i.e. my-file.bw.cg, my-file.bw.chg, and my-file.bw.chh.
    • Visualization greatly depends on if the -all parameter is used when converting the raw allC/bismark file

File Conversion

The methylation tracks works best when the data is stored as BigWig file(s). File conversion is easy, though. Use the conversion program appropriate to your input file type. bedGraphToBigWig and bedSort (programs from UCSC) must be on your path.

allC Files

  • allC files have these columns: chr, pos, strand, mc_class, mc_count, total, methylated

  • In v2, by default, only methylated positions are included. Use the -all option to include all non-zero positions.

  • All chromosomes need to be combined into one allC file.

  • You also need a file with chromosome sizes. This file should be tab-delimitated, one chromosome per line, with at least chromosome name and chromosome size. The genome's FASTA index (.fa.fai) file works well.

  • For a given genome (i.e. chromosome sizes), you can specify an unlimited number of allC files to be converted.

  • Note: version 1 conversion renames reference sequences to a convention while version 2 does not.

  • Conversion Version 1:

Usage: python3 allc_old_bigwig_pe.py [-keep] [-no-clean] [-sort] [-all] 
       [-L=labels] [-p=num_proc] <chrm_sizes>  <allC_file> [allC_file]*  
-keep           when on, keeps temporary files
-no-clean       does not check chromosome names match chrm file
-sort           calls bedSort; add this option if bigwig conversion fails
-all            use all positions with methylation not just methylated ones
-L=labels       comma-separated list of labels for incoming allC files  
-p=num_proc     number of processors to use [default 1]  
-o=outID        optional string to include in output file names
  • Conversion Version 2:
Usage: python3 allc_to_bigwig_pe.py [-keep] [-sort] [-all] [-L=labels] 
       [-o=out_id] [-p=num_proc]<chrm_sizes>  <allC_file> [allC_file]*
-keep        keep intermediate files
-sort        calls bedSort; add this option if bigwig conversion fails
-all         use all positions with methylation [default methylated sites]
-allz        use all positions with coverage including 0's
-L=labels    comma-separated list of labels to use for the allC files;
             defaults to using information from the allc file name
-o=out_id    optional identifier to be added to the output file names
-p=num_proc  number of processors to use [default 1]

Bismark

  • Bismark files have the following columns: chr, pos, strand, methylated reads, total reads, C context, trinucleotide context

  • There is a script to convert bismark files to version 2

  • Note: for v3, when filtering for methylated sites only, any site with at least 1 methylated read is considered "methylated"

  • Conversion Version 2

Usage:  python3 bismark_to_bigwig_pe.py [-keep] [-sort] [-all] [-L=labels] 
        [-o=out_id] [-p=num_proc] <chrm_sizes>  <bismark_file> [bismark_file]*
-keep        keep intermediate files
-sort        calls bedSort; add this option if bigwig conversion fails
-all         use all covered positions including 0s [default only includes mC > 1]
-L=labels    comma-separated list of labels to use for the allC files;
             defaults to using information from the allc file name
-o=out_id    optional identifier to be added to the output file names
-p=num_proc  number of processors to use [default 1]

JSON Track Specifications

Track specifications are very similar to those for XYPlots (see JBrowse tutorial for more information). The label, type, and urlTemplate must be specified.

Version 1

{  
  "key" : "Wild Type Methylation",
  "label" : "track_wild_type_methylation",
  "style" : { "height" : 50 },
  "storeClass" : "JBrowse/Store/SeqFeature/BigWig",
  "urlTemplate" : "path/to/bigwig_file.bw",
  "type" : "MethylationPlugin/View/Track/Wiggle/MethylXYPlot"
}

Version 2

urlTemplate is the path and filename up-to, but not including, the context-specific extension. For example "urlTemplate" : "path/my-file.bw".

{  
  "key" : "Wild Type Methylation",
  "label" : "track_wild_type_methylation",
  "style" : { "height" : 50 },
  "methylatedOption" : false,
  "storeClass" : "MethylationPlugin/Store/SeqFeature/MethylBigWig",
  "urlTemplate" : "path/to/bigwig_file.bw",
  "type" : "MethylationPlugin/View/Track/Wiggle/MethylPlot"
}

Same as Version 3 except

"methylatedOption" : false