Question: Error message by dba.plotBox() function
gravatar for xie186
9 weeks ago by
xie1860 wrote:

When I was using DiffBind, I got the error message below from  dba.plotBox(): 

Error in wilcox.test.default(toplot[[i]], toplot[[j]], paired = FALSE) :

  not enough 'y' observations

Calls: runDiffBind ... pv.plotBoxplot -> pvalMethod -> wilcox.test.default

Could someone tell me the possible reason of this? Thanks in advance. 

The code is shown as below:

    samples = read.csv(input)

    # read the peaks into a DBA object.

    # 1) plot
    # by default, it will plots a correlation heatmap
    #Correlation heatmap using occupacy (peak caller score) data
    #For the peaks of SICER, I used fold change values here
    tamoxifen = dba(sampleSheet=input)

    #calculate a binding matrix with cores based on read counts for every sample.
    # 2) plot
    # correlation heatmap using read count data by default
    tamoxifen = dba.count(tamoxifen, minOverlap=3, bRemoveDuplicates = FALSE)

    # Establishing a contrast.
    tamoxifen<-dba.contrast(tamoxifen, minMembers=2)
    # dba.analyze is the main differential analysis function
    # 3) plot
    # by default, it will plots a correlation heatmap if diff peaks are found
    # correlation heatmap using read count data of diff peaks.
    cat("-dba.analyze", "\n")
    tamoxifen<-dba.analyze(tamoxifen, bSubControl = FALSE)

    cat("", "\n")
    tamoxifen.DB<, th = thrd)

    # MA plot of comparison: Sites identified as significantly differentially bound shown in red.
    # 4) plot:
    cat("dba.plotMA", "\n")

    cat("dba.fold", "\n")


    # 4) plot PCA
    cat("dba.plotPCA", "\n")
    dba.plotPCA(tamoxifen, label = DBA_REPLICATE)

    cat("dba.plotPCA", "\n")
    # 5) plot PCA using count data for only differentailly bound sites.
    dba.plotPCA(tamoxifen, contrast=1, th = thrd, label = DBA_REPLICATE)

    #cat("dba.plotBox", "\n")
    # 6) Boxplot of read distribution for differential peaks

    cat("dba.plotHeatmap", "\n")
    # 7) Heatmap showing binding affinities for differentially peaks
    dba.plotHeatmap(tamoxifen, contrast=1, correlations=FALSE)

    ## we need elementMetadata to
     cat("Write the table", "\n")
     df <- data.frame(seqnames=seqnames(tamoxifen.DB), starts=start(tamoxifen.DB),  ends=end(tamoxifen.DB),elementMetadata(tamoxifen.DB))
    write.table(df, file=out, quote=F, sep="\t", row.names = F)

Here is the session information:

> sessionInfo()
R version 3.3.0 (2016-05-03)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Red Hat Enterprise Linux Server release 6.9 (Santiago)

 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C
 [9] LC_ADDRESS=C               LC_TELEPHONE=C

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

loaded via a namespace (and not attached):
 [1] Biobase_2.32.0             edgeR_3.14.0
 [3] bit64_0.9-7                splines_3.3.0
 [5] gtools_3.5.0               assertthat_0.2.0
 [7] stats4_3.3.0               latticeExtra_0.6-28
 [9] amap_0.8-14                RBGL_1.48.1
[11] blob_1.1.0                 Rsamtools_1.24.0
[13] Category_2.38.0            RSQLite_2.0
[15] backports_1.1.0            lattice_0.20-35
[17] glue_1.1.1                 limma_3.28.21
[19] digest_0.6.12              GenomicRanges_1.24.3
[21] RColorBrewer_1.1-2         XVector_0.12.1
[23] checkmate_1.7.4            colorspace_1.3-2
[25] Matrix_1.2-6               plyr_1.8.4
[27] GSEABase_1.34.1            XML_3.98-1.9
[29] pkgconfig_2.0.1            pheatmap_1.0.8
[31] ShortRead_1.30.0           biomaRt_2.28.0
[33] genefilter_1.54.2          zlibbioc_1.18.0
[35] xtable_1.8-2               GO.db_3.3.0
[37] scales_0.5.0               brew_1.0-6
[39] gdata_2.18.0               BiocParallel_1.6.6
[41] tibble_1.3.4               annotate_1.50.1
[43] IRanges_2.6.1              ggplot2_2.2.1.9000
[45] SummarizedExperiment_1.2.3 GenomicFeatures_1.24.2
[47] BiocGenerics_0.18.0        lazyeval_0.2.0
[49] survival_2.41-3            magrittr_1.5
[51] memoise_1.1.0              systemPipeR_1.6.2
[53] fail_1.3                   gplots_3.0.1
[55] hwriter_1.3.2              DiffBind_2.0.2
[57] GOstats_2.38.0             graph_1.50.0
[59] tools_3.3.0                BBmisc_1.9
[61] stringr_1.2.0              sendmailR_1.2-1
[63] S4Vectors_0.10.3           munsell_0.4.3
[65] locfit_1.5-9.1             bindrcpp_0.2
[67] AnnotationDbi_1.34.4       Biostrings_2.40.2
[69] GenomeInfoDb_1.8.7         caTools_1.17.1
[71] rlang_0.1.2                grid_3.3.0
[73] RCurl_1.95-4.8             rjson_0.2.15
[75] AnnotationForge_1.14.2     bitops_1.0-6
[77] base64enc_0.1-3            gtable_0.2.0
[79] DBI_0.7                    R6_2.2.2
[81] GenomicAlignments_1.8.4    dplyr_0.7.2
[83] rtracklayer_1.32.0         bit_1.1-12
[85] bindr_0.1                  KernSmooth_2.23-15
[87] stringi_1.1.5              parallel_3.3.0
[89] BatchJobs_1.6              Rcpp_0.12.12
ADD COMMENTlink modified 9 weeks ago by Rory Stark2.1k • written 9 weeks ago by xie1860
gravatar for Rory Stark
9 weeks ago by
Rory Stark2.1k
CRUK, Cambridge, UK
Rory Stark2.1k wrote:


I tried running your script with the samplesheet and data from the vignette and it works fine. This is using the current version of DiffBind (2.4), I see that you are using a somewhat older version. (Also, I wasn't sure what the value of the variable thrd is, I used thrd <- 3, but it doesn't really matter as your call to dba.plotBox() did not specify a contrast and hence uses the default contrast=1.)

If you could email me your DBA object tamoxifen I can try the call to see if I can reproduce the problem with the current released version of DiffBind.



ADD COMMENTlink written 9 weeks ago by Rory Stark2.1k

Thanks for your reply. thrd is the threshold. I tried the latest version and I still got the message. 

I sent you an email with the DBA object tamoxifen. 

<font face="monospace">Shaojun </font>

ADD REPLYlink written 9 weeks ago by xie1860

OK, I see what's going on. All of the differentially bound sites are + (fold change > 0; "gained"), and there are zero - ("loss") sites. DiffBind  generates the plot just fine, but is failing to detect this condition properly and generates an error when computing the p-values of the differences.

You can turn off the calculation of the p-values by setting pvalMethod=NULL, however there is another bug that will generate a different error. I'll fix both these issues soon. In the mean time, you are actually getting the plot, if not the p-values (which aren't very interesting). With all the differences occurring in one direction (seen in dba.polotMA(), or checking the signs of the fold changes in the report), the box plot isn't very illuminating.



ADD REPLYlink written 9 weeks ago by Rory Stark2.1k

Thank you. 

ADD REPLYlink written 9 weeks ago by xie1860
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