Help me to find out the reason of this error in RnBeads...

Question

[miniymay101]

Hello,

I am a beginner who start to study bioinformatics.

Now I am trying to run RnBeads (R package) to analyze bisulfite sequencing data. I have beed used this package for several times and had successful results without any error.

But this time I tried for 3 times, but still getting an same error, and cannot guess what the problem is.

Is there any one who can help me to solve or fix this error?

I used "bismarkcov" format files, which are results of bismark and want to get result of differential methylation analysis.

My code is like this :

rnb.options(
identifiers.column="sampleID",
import.bed.style="bismarkCov",
assembly="hg38",
differential.report.sites=FALSE
)

report.dir <- file.path(getwd(), "results_diffMethyl_3")

data.dir <- file.path(getwd())

sample.annotation <- file.path(getwd(), "sample_annotation.csv")

data.source <- c(data.dir, sample.annotation)

rnb.set <- rnb.execute.import(data.source=data.source, data.type="bs.bed.dir")

rnb.set.unfiltered <- rnb.set

result_preprocessing <- rnb.run.preprocessing(rnb.set.unfiltered, dir.reports=report.dir)

rnb2.set <- result_preprocessing$rnb.set

rnb.run.differential(rnb2.set, report.dir)

And the error is like...

2018-01-17 23:38:37    11.2  STATUS                             COMPLETED Adding volcano plots

2018-01-17 23:38:37    11.2  STATUS                             STARTED Adding tables

Error in data.frame(..., check.names = FALSE) :

  arguments imply differing number of rows: 583340, 218725

In addition: There were 12 warnings (use warnings() to see them)

I would be really grateful if you help to find out the reason or fix this error...

I am working on this for 7days...

[WouterDeCoster]

I added code markup to your post for increased readability. You can do this by selecting the text and clicking the 101010 button. When you compose or edit a post that button is in your toolbar, see image below:

[Friederike]

can you show the result of str(rnb2.set)?

[miniymay101]

> str(rnb2.set)
Formal class 'RnBiseqSet' [package "RnBeads"] with 12 slots
  ..@ status             :List of 2
  .. ..$ disk.dump      : logi TRUE
  .. ..$ disk.dump.bigff: logi FALSE
  ..@ pheno              :'data.frame': 18 obs. of  3 variables:
  .. ..$ sampleID    : Factor w/ 18 levels "Bcell_1000_D1_R1",..: 2 3 1 5 6 4 8 9 7 11 ...
  .. ..$ filename_bed: Factor w/ 18 levels "GSM2324434_Bcell_1000_D2_R1.cov",..: 1 2 3 4 5 6 7 8 9 10 ...
  .. ..$ cell_type   : Factor w/ 6 levels "B_cell","CD4",..: 1 1 1 2 2 2 3 3 3 4 ...
  ..@ sites              : num [1:27865223, 1:3] 1 1 1 1 1 1 1 1 1 1 ...
  .. ..- attr(*, "dimnames")=List of 2
  .. .. ..$ : NULL
  .. .. ..$ : chr [1:3] "" "" "regInd"
  ..@ meth.sites         : list()
  .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. ..- attr(*, "vmode")= chr "double"
  .. .. ..- attr(*, "maxlength")= int 501574014
  .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a574e9ac280.ff"
  .. .. ..- attr(*, "pagesize")= int 65536
  .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. ..- attr(*, "virtual")= list()
  .. .. ..- attr(*, "Length")= int 501574014
  .. .. ..- attr(*, "Dim")= int [1:2] 27865223 18
  .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. ..$ : NULL
  .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
 .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"
  ..@ covg.sites         : list()
  .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. ..- attr(*, "vmode")= chr "integer"
  .. .. ..- attr(*, "maxlength")= int 501574014
  .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a57465c2735.ff"
  .. .. ..- attr(*, "pagesize")= int 65536
  .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. ..- attr(*, "virtual")= list()
  .. .. ..- attr(*, "Length")= int 501574014
  .. .. ..- attr(*, "Dim")= int [1:2] 27865223 18
  .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. ..$ : NULL
  .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
 .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"

[miniymay101]

  ..@ regions            :List of 4
  .. ..$ tiling    : num [1:583340, 1:3] 1 1 1 1 1 1 1 1 1 1 ...
  .. .. ..- attr(*, "dimnames")=List of 2
  .. .. .. ..$ : NULL
  .. .. .. ..$ : chr [1:3] "" "" "regInd"
  .. ..$ genes     : num [1:55774, 1:3] 1 1 1 1 1 1 1 1 1 1 ...
  .. .. ..- attr(*, "dimnames")=List of 2
  .. .. .. ..$ : NULL
  .. .. .. ..$ : chr [1:3] "" "" "regInd"
  .. ..$ promoters : num [1:59451, 1:3] 1 1 1 1 1 1 1 1 1 1 ...
  .. .. ..- attr(*, "dimnames")=List of 2
  .. .. .. ..$ : NULL
  .. .. .. ..$ : chr [1:3] "" "" "regInd"
  .. ..$ cpgislands: num [1:27541, 1:3] 1 1 1 1 1 1 1 1 1 1 ...
  .. .. ..- attr(*, "dimnames")=List of 2
  .. .. .. ..$ : NULL
  .. .. .. ..$ : chr [1:3] "" "" "regInd"
  ..@ meth.regions       :List of 4
  .. ..$ tiling    : list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "double"
  .. .. .. ..- attr(*, "maxlength")= int 10500120
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a576fd3fec0.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 10500120
  .. .. .. ..- attr(*, "Dim")= int [1:2] 583340 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
  .. .. ..- attr(*, "aggregation")= Named chr "mean"
  .. .. .. ..- attr(*, "names")= chr "tiling"
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"

[miniymay101]

  .. ..$ genes     : list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "double"
  .. .. .. ..- attr(*, "maxlength")= int 1003932
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a5740f6c639.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 1003932
  .. .. .. ..- attr(*, "Dim")= int [1:2] 55774 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
  .. .. ..- attr(*, "aggregation")= Named chr "mean"
  .. .. .. ..- attr(*, "names")= chr "genes"
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"
  .. ..$ promoters : list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "double"
  .. .. .. ..- attr(*, "maxlength")= int 1070118
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a57298541b1.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 1070118
  .. .. .. ..- attr(*, "Dim")= int [1:2] 59451 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
  .. .. ..- attr(*, "aggregation")= Named chr "mean"
  .. .. .. ..- attr(*, "names")= chr "promoters"
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"
  .. ..$ cpgislands: list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "double"
  .. .. .. ..- attr(*, "maxlength")= int 495738
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a576d890e62.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 495738
  .. .. .. ..- attr(*, "Dim")= int [1:2] 27541 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
  .. .. ..- attr(*, "aggregation")= Named chr "mean"
  .. .. .. ..- attr(*, "names")= chr "cpgislands"
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"

[miniymay101]

 ..@ covg.regions       :List of 4
  .. ..$ tiling    : list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "integer"
  .. .. .. ..- attr(*, "maxlength")= int 10500120
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a57726f73fb.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 10500120
  .. .. .. ..- attr(*, "Dim")= int [1:2] 583340 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"
  .. ..$ genes     : list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "integer"
  .. .. .. ..- attr(*, "maxlength")= int 1003932
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a574b9c79dd.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 1003932
  .. .. .. ..- attr(*, "Dim")= int [1:2] 55774 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"
  .. ..$ promoters : list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "integer"
  .. .. .. ..- attr(*, "maxlength")= int 1070118
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a57587e7bc.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 1070118
  .. .. .. ..- attr(*, "Dim")= int [1:2] 59451 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"
  .. ..$ cpgislands: list()
  .. .. ..- attr(*, "physical")=Class 'ff_pointer' <externalptr> 
  .. .. .. ..- attr(*, "vmode")= chr "integer"
  .. .. .. ..- attr(*, "maxlength")= int 495738
  .. .. .. ..- attr(*, "pattern")= chr "ff"
  .. .. .. ..- attr(*, "filename")= chr "/data1/project/Xeno/project_jm/tempfiles/Rtmp2dRiI8/ff7a57e4f5554.ff"
  .. .. .. ..- attr(*, "pagesize")= int 65536
  .. .. .. ..- attr(*, "finalizer")= chr "delete"
  .. .. .. ..- attr(*, "finonexit")= logi TRUE
  .. .. .. ..- attr(*, "readonly")= logi FALSE
  .. .. .. ..- attr(*, "caching")= chr "mmnoflush"
  .. .. ..- attr(*, "virtual")= list()
  .. .. .. ..- attr(*, "Length")= int 495738
  .. .. .. ..- attr(*, "Dim")= int [1:2] 27541 18
  .. .. .. ..- attr(*, "Dimorder")= int [1:2] 1 2
  .. .. .. ..- attr(*, "Symmetric")= logi FALSE
  .. .. .. ..- attr(*, "Dimnames")=List of 2
  .. .. .. .. ..$ : NULL
  .. .. .. .. ..$ : chr [1:18] "Bcell_1000_D2_R1" "Bcell_1000_D3_R1" "Bcell_1000_D1_R1" "CD4_1000_D2_R1" ...
 .. .. .. - attr(*, "class") =  chr [1:3] "ff_matrix" "ff_array" "ff"
  ..@ assembly           : chr "hg38"
  ..@ target             : chr "CpG"
  ..@ inferred.covariates: list()
  ..@ version            : chr "1.6.1"

Thank you for giving me a comment! I would be very appreciate if you keep helping me to solve the problem..

[Friederike]

in your working directory, do you have any results from the differential analysis? I'm just trying to understand at what point it's failing

[miniymay101]

In differential methylation analysis html page, I got regional level - scatter plots & volcano plots, but "Differential Methylation Tables" subsection is totally missing.

It only has :

    A tabular overview of measures for differential methylation on the region level for the individual comparisons are provided in this section.

id: region id
Chromosome: chromosome of the region
Start: start coordinate of the region
End: end coordinate of the region
[symbol]: associated gene symbol to the given region [only valid for gene associated regions]
[entrezID]: Entrez ID of the gene associated with the region [only valid for gene associated regions]
mean.mean.g1,mean.mean.g2: (where g1 and g2 is replaced by the respective group names in the table) mean of mean methylation levels for group 1 and 2 across all sites in a region
mean.mean.diff: Mean difference in means across all sites in a region
mean.mean.quot.log2: log2 of the mean quotient in means across all sites in a region
comb.p.val: Combined p-value aggregating p-values of all sites in the region using a generalization of Fisher's method [1]
comb.p.adj.fdr: FDR adjusted combined p-value
combinedRank: mean.mean.diff, mean.mean.quot.log2 and comb.p.val are ranked for all regions. This column aggregates them using the maximum, i.e. worst rank of a site among the three measures
num.sites: number of sites associated with the region
mean.num.na.g1,mean.num.na.g2: Mean number of NA methylation values accross all sites in group 1 and group 2 respectively
mean.mean.covg.g1,mean.mean.covg.g2: Mean value of mean coverage values (across all samples in a group) across all sites in a region
mean.nsamples.covg.thresh.g1,mean.nsamples.covg.thresh.g2: mean number of samples (accross all considered sites) that have a coverage larger than 5 for the site in group 1 and group 2 respectively
The tables for the individual comparisons can be found here:

and after this, there is nothing...

Answer 1

[fmueller]

Hi, sorry for being late to the game. If you shoot us an email (to [e-mail]) we are typically faster. The problem you describe occurs sometimes when the disk-based files that we use to store the (differential) methylation data (using the ff package) are corrupted. In our experience this happens sometimes, e.g. when the I/O on the filesystem is high. Typically rerunning the analysis helps in resolving this issue. Hope that helps.