DiffBind -- Error: No sites have activity greater than minMaxval
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Entering edit mode
Alexandre • 0
@095e334e
Last seen 2 days ago
Hong Kong

Enter the body of text here

When running dba.count() on my dba object:

samples <- dba(sampleSheet = "../samplesheet.csv",peakFormat = "bed",scoreCol = 5)

ep_293T_siNeg_R1  ep_293T_siNeg   1 bed ep_293T_siNeg_R2  ep_293T_siNeg   2 bed ep_293T_siFUS_R1  ep_293T_siFUS   1 bed ep_293T_siFUS_R2  ep_293T_siFUS   2 bed

I get the following error:

samples <- dba.count(samples)

Computing summits...
Sample: shard-0/execution/10249_sample1.bam125 
Sample: shard-1/execution/10249_sample2.bam125 
Sample: shard-2/execution/10249_sample3.bam125 
Sample: shard-3/execution/10249_sample4.bam125 
Sample: shard-4/execution/10249_sample5.bam125 
Sample: shard-5/execution/10249_sample6.bam125 
Sample: shard-6/execution/10249_sample7.bam125 
Sample: shard-7/execution/10249_sample8.bam125 
Re-centering peaks...
Sample: shard-0/execution/10249_sample1.bam125 
Reads will be counted as Paired-end.
Sample: shard-1/execution/10249_sample2.bam125 
Reads will be counted as Paired-end.
Sample: shard-2/execution/10249_sample3.bam125 
Reads will be counted as Paired-end.
Sample: shard-3/execution/10249_sample4.bam125 
Reads will be counted as Paired-end.
Sample: shard-4/execution/10249_sample5.bam125 
Reads will be counted as Paired-end.
Sample: shard-5/execution/10249_sample6.bam125 
Reads will be counted as Paired-end.
Sample: shard-6/execution/10249_sample7.bam125 
Reads will be counted as Paired-end.
Sample: shard-7/execution/10249_sample8.bam125 
Reads will be counted as Paired-end.
Error: No sites have activity greater than minMaxval
In addition: Warning message:
Parallel execution unavailable: executing serially.

What am I getting wrong here ? Thanks. I noticed there a strange number concatenated to the end of my .bam files i.e 125.

P.S: from github the error stems from this section : https://github.com/aeron15/DiffBind/blob/master/R/counts.R#L579 but I'm not familiar with what those lines mean.

R version 4.1.0 (2021-05-18)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)

Matrix products: default

locale:
[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252    LC_MONETARY=English_United States.1252 LC_NUMERIC=C                          
[5] LC_TIME=English_United States.1252    

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

other attached packages:
 [1] DiffBind_3.2.1              SummarizedExperiment_1.22.0 Biobase_2.52.0              MatrixGenerics_1.4.0        matrixStats_0.58.0         
 [6] GenomicRanges_1.44.0        GenomeInfoDb_1.28.0         IRanges_2.26.0              S4Vectors_0.30.0            BiocGenerics_0.38.0        

loaded via a namespace (and not attached):
  [1] backports_1.2.1          GOstats_2.58.0           BiocFileCache_2.0.0      plyr_1.8.6               GSEABase_1.54.0          splines_4.1.0           
  [7] BiocParallel_1.26.0      ggplot2_3.3.3            amap_0.8-18              digest_0.6.27            invgamma_1.1             GO.db_3.13.0            
 [13] SQUAREM_2021.1           fansi_0.4.2              magrittr_2.0.1           checkmate_2.0.0          memoise_2.0.0            BSgenome_1.60.0         
 [19] base64url_1.4            limma_3.48.0             Biostrings_2.60.0        annotate_1.70.0          systemPipeR_1.26.2       bdsmatrix_1.3-4         
 [25] prettyunits_1.1.1        jpeg_0.1-8.1             colorspace_2.0-1         blob_1.2.1               rappdirs_0.3.3           apeglm_1.14.0           
 [31] ggrepel_0.9.1            dplyr_1.0.6              crayon_1.4.1             RCurl_1.98-1.3           jsonlite_1.7.2           graph_1.70.0            
 [37] genefilter_1.74.0        brew_1.0-6               survival_3.2-11          VariantAnnotation_1.38.0 glue_1.4.2               gtable_0.3.0            
 [43] zlibbioc_1.38.0          XVector_0.32.0           DelayedArray_0.18.0      V8_3.4.2                 Rgraphviz_2.36.0         scales_1.1.1            
 [49] pheatmap_1.0.12          mvtnorm_1.1-1            DBI_1.1.1                edgeR_3.34.0             Rcpp_1.0.6               xtable_1.8-4            
 [55] progress_1.2.2           emdbook_1.3.12           bit_4.0.4                rsvg_2.1.2               AnnotationForge_1.34.0   truncnorm_1.0-8         
 [61] httr_1.4.2               gplots_3.1.1             RColorBrewer_1.1-2       ellipsis_0.3.2           pkgconfig_2.0.3          XML_3.99-0.6            
 [67] dbplyr_2.1.1             locfit_1.5-9.4           utf8_1.2.1               tidyselect_1.1.1         rlang_0.4.11             AnnotationDbi_1.54.0    
 [73] munsell_0.5.0            tools_4.1.0              cachem_1.0.5             generics_0.1.0           RSQLite_2.2.7            stringr_1.4.0           
 [79] fastmap_1.1.0            yaml_2.2.1               bit64_4.0.5              caTools_1.18.2           purrr_0.3.4              KEGGREST_1.32.0         
 [85] RBGL_1.68.0              biomaRt_2.48.0           compiler_4.1.0           rstudioapi_0.13          filelock_1.0.2           curl_4.3.1              
 [91] png_0.1-7                tibble_3.1.2             stringi_1.6.1            GenomicFeatures_1.44.0   lattice_0.20-44          Matrix_1.3-3            
 [97] vctrs_0.3.8              pillar_1.6.1             lifecycle_1.0.0          data.table_1.14.0        bitops_1.0-7             irlba_2.3.3             
[103] rtracklayer_1.52.0       R6_2.5.0                 BiocIO_1.2.0             latticeExtra_0.6-29      hwriter_1.3.2            ShortRead_1.50.0        
[109] KernSmooth_2.23-20       MASS_7.3-54              gtools_3.8.2             assertthat_0.2.1         Category_2.58.0          rjson_0.2.20            
[115] withr_2.4.2              GenomicAlignments_1.28.0 batchtools_0.9.15        Rsamtools_2.8.0          GenomeInfoDbData_1.2.6   hms_1.1.0               
[121] grid_4.1.0               DOT_0.1                  coda_0.19-4              GreyListChIP_1.24.0      ashr_2.2-47              mixsqp_0.3-43
DiffBind • 310 views
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Entering edit mode
Rory Stark ★ 4.1k
@rory-stark-5741
Last seen 9 hours ago
CRUK, Cambridge, UK

The error message need fixing as it refers to an internal value(minMaxval) instead of the parameter filter.

This is saying that all the computed RPKM values are less than the default filter value of 1.0.

You may want to check to see what the matrix looks like after you read in the experiment and before counting. You can see it as follows:

dba.peakset(samples, bRetrieve=TRUE)

This way to can check that you have a set of intervals where you expect to find uniquely overlapping reads.

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thanks Rory, I've got this matrix after calling dba.peakset(samples) , I guess there is something odd happening when reading my sampleSheet, since I have defined only 4 samples not 5.

5 Samples, 546816 sites in matrix (2979829 total):
                ID Condition Replicate Caller Intervals Reads
1 ep_293T_siNeg_R1     siNeg         1    bed    915009    NA
2 ep_293T_siNeg_R2     siNeg         1    bed   1212181    NA
3 ep_293T_siFUS_R1     siFUS         2    bed   1113536    NA
4 ep_293T_siFUS_R2     siFUS         2    bed   1836982    NA
5                5                                0        0

My samplesheet is the following:

SampleID    Tissue  Factor  Condition   Treatment   Replicate   bamReads    ControlID   bamControl  Peaks   PeakCaller
ep_293T_siNeg_R1    NA  NA  siNeg   NA  1   shard-0/execution/10249_sample1.bam input_293T_siNeg_R1 shard-4/execution/10249_sample5.bam 10249_sample1.bed   bed
ep_293T_siNeg_R2    NA  NA  siNeg   NA  1   shard-1/execution/10249_sample2.bam input_293T_siNeg_R2 shard-5/execution/10249_sample6.bam 10249_sample2.bed   bed
ep_293T_siFUS_R1    NA  NA  siFUS   NA  2   shard-2/execution/10249_sample3.bam input_293T_siFUS_R1 shard-6/execution/10249_sample7.bam 10249_sample3.bed   bed
ep_293T_siFUS_R2    NA  NA  siFUS   NA  2   shard-3/execution/10249_sample4.bam input_293T_siFUS_R2 shard-7/execution/10249_sample8.bam 10249_sample4.bed   bed
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I should have included bRetrieve=TRUE in the call to dba.peakset() in my original answer-- I have edited the answer to show the correct code:

dba.peakset(samples, bRetrieve=TRUE)

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was this error resolved? i now have the same error, including when running files that previously ran successfully. thank you.

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Nicole, are you getting the original Error: No sites have activity greater than minMaxval error, or having a problem after calling dba.peakset() to view the count matrix? If it is the latter, see the correction above, the call should be:

dba.peakset(samples, bRetrieve=TRUE)

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Actually I solved my problem by passing in the flag bUseSummarizeOverlaps = F in samples <- dba.count(samples,bUseSummarizeOverlaps = F). Hopefully it's not too different of a count method than the default one.

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If the reads are single end, it is very close; for paired-end reads, each read is counted separately. The read counts end up double but the calculation of differential binding is consistent. If there were some problem with summarizeOverlaps finding matching reads in what should be a paired-end run, the alternative method work well.

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