Heatmap/Violin plot issues using amplicons as regions

[sdewell]

Hello,

Are there any known issues using a set of amplicon coordinates as a reference for the Heatmap functions? I'm using three amplicons as the 'genome' to which I've aligned, and most analyses are working well but when running the heatmap function I receive an error:

Error in sample.int(length(x), size, replace, prob) : cannot take a sample larger than the population when 'replace = FALSE' Calls: tryCatch ... tryCatchList -> tryCatchOne -> -> sample -> sample.int Execution halted

I realize it's an R error but not sure how to fix the data structure issues feeding into the R script. There are 22 samples, and three amplicons ~100-175bp. The violin plots are not being created as well. I've previously used a whole genome reference, and then intersected the .cov files with my coordinates for the bis_rep files; this works but is rather slow and takes a lot of disk space for large sample numbers.

Thanks for any suggestions!

[readbio]

Hi, can you provide the table generated by ViewBS? Then I can test the R code and fix the problem. Thanks.

[sdewell]

Here's my command and output: /usr/bin/ViewBS/ViewBS MethHeatmap --verbose --region ../amplicons.bed --sample 01-trimmed_bismark.bis_rep.cov.gz,01 --sample 02-trimmed_bismark.bis_rep.cov.gz,02 --sample 03-trimmed_bismark.bis_rep.cov.gz,03 --sample 04-trimmed_bismark.bis_rep.cov.gz,04 --sample 05-trimmed_bismark.bis_rep.cov.gz,05 --sample 06-trimmed_bismark.bis_rep.cov.gz,06 --sample 07-trimmed_bismark.bis_rep.cov.gz,07 --outdir MethHeatmap --context CHG --prefix CHG

Subcommand: MethHeatmap 01-trimmed_bismark.bis_rep.cov.gz,01 02-trimmed_bismark.bis_rep.cov.gz,02 03-trimmed_bismark.bis_rep.cov.gz,03 04-trimmed_bismark.bis_rep.cov.gz,04 05-trimmed_bismark.bis_rep.cov.gz,05 06-trimmed_bismark.bis_rep.cov.gz,06 07-trimmed_bismark.bis_rep.cov.gz,07 ../amplicons.bed Output directory is: MethHeatmap Output prefix: CHG Start calculate methylation information for target context CHG Error in sample.int(length(x), size, replace, prob) : cannot take a sample larger than the population when 'replace = FALSE' Calls: tryCatch ... tryCatchList -> tryCatchOne -> -> sample -> sample.int Execution halted Meth::Heatmap=HASH(0x1957810): WARNING: unknown option '--input'

ARGUMENT '/home/scottd/projects/methylation_apr_2017/trim_galore/combined/amplicons_ref/MethHeatmap/CHG_MethHeatmap_CHG.txt' ignored

WARNING: unknown option '--output1'

ARGUMENT '/home/scottd/projects/methylation_apr_2017/trim_galore/combined/amplicons_ref/MethHeatmap/CHG_MethHeatmap_CHG.pdf' ignored

WARNING: unknown option '--output2'

ARGUMENT '/home/scottd/projects/methylation_apr_2017/trim_galore/combined/amplicons_ref/MethHeatmap/CHG_MethHist_CHG.pdf' ignored

WARNING: unknown option '--cluster_cols'

ARGUMENT 'FALSE' ignored

WARNING: unknown option '--cluster_rows'

ARGUMENT 'TRUE' ignored

WARNING: unknown option '--height'

ARGUMENT '10' ignored

WARNING: unknown option '--width'

ARGUMENT '10' ignored

WARNING: unknown option '--height2'

ARGUMENT '10' ignored

WARNING: unknown option '--width2'

ARGUMENT '10' ignored

WARNING: unknown option '--random_region'

ARGUMENT '20' ignored

Useage: R --vanilla --slave --input <> --height --width --height2 --width2 --random_region --output1 --output2 < trans_DMS_heatmap.R /usr/lib/R/bin/exec/R --vanilla --slave --input /home/scottd/projects/methylation_apr_2017/trim_galore/combined/amplicons_ref/MethHeatmap/CHG_MethHeatmap_CHG.txt --output1 /home/scottd/projects/methylation_apr_2017/trim_galore/combined/amplicons_ref/MethHeatmap/CHG_MethHeatmap_CHG.pdf --output2 /home/scottd/projects/methylation_apr_2017/trim_galore/combined/amplicons_ref/MethHeatmap/CHG_MethHist_CHG.pdf --cluster_cols FALSE --cluster_rows TRUE --height 10 --width 10 --height2 10 --width2 10 --random_region 20 FALSE TRUE

Running time: 0 wallclock secs ( 0.01 usr 0.00 sys + 0.29 cusr 0.03 csys = 0.33 CPU)

The txt file is all NA 01 02 03 04 05 06 07 chr19_58552755_58552920 NA NA NA NA NA NA NA chr11_119089504_119089643 NA NA NA NA NA NA NA chr22_24572821_24572937 NA NA NA NA NA NA NA

Example lines from bgzipped/tabix'ed cov file: chr19 10 - 64 40 CHG CTG chr19 11 + 18988 1 CHH CCA chr19 12 + 20073 7 CHH CAT chr19 16 + 22070 7 CHH CCT chr19 17 + 21995 49 CHH CTC

My bed file is standard and workd for other analyses. There are reads/methylation information outside of the bed coordinates, but that shouldn't be a problem. Appreciate your help and thanks for the tool!

[readbio]

Thanks for your information. Since I can't repeat your command line, I can just guess the possible reason.

One possibility is that ViewBS MethHeatmap will exclude the sites with read depth < 5 by default. So if you use the default parameters and all the read depths of the given regions are below 5, no information will be return. So maybe what you can do is set "--minDepth" as 1 and try again.

See the corresponding code below:

   if(!$opts_sub->{"minDepth"}){
        $opts_sub->{"minDepth"} = 5;
    }

Please let me know if this works or not.

[sdewell]

Thank you - the minDepth helped, though I had also made an error in my bismark cov file that was compounding the errors. Appreciate the responses!

[readbio]

Glad to hear that you fix your problem. Please let us know if you have any questions or suggestions. These can help us to improve ViewBS in the future. Thanks.