DiffBind analysis for ATAC-Seq error Error processing one or more read files
1
0
Entering edit mode
Asma • 0
@asma-24876
Last seen 6 months ago

I want to find differential list of genomic sites that are unique and shared between the the 3 treg conditions (WT, ON, OFF) with 2 replicates per group (TREG-WT-1, TREG-WT-2; TREG-ON-1, TREG-ON-2; TREG-OFF-1, TREG-OFF-2) .

Here is the sample sheet provided:

11_Treg,WT,1,11_Treg_S7_L001_R1_001.trim.merged.bam,11_Treg_S7_L001_R1_001.trim.merged.nodup.no_chrM_MT.tn5.pval0.01.300K.bfilt.narrowPeak.gz,narrow 12_Treg,WT,2,12_Treg_S8_L001_R1_001.trim.merged.bam,12_Treg_S8_L001_R1_001.trim.merged.nodup.no_chrM_MT.tn5.pval0.01.300K.bfilt.narrowPeak.gz,narrow 51F_Treg,ON,1,51F_Treg_S9_L001_R1_001.trim.merged.bam,51F_Treg_S9_L001_R1_001.trim.merged.nodup.no_chrM_MT.tn5.pval0.01.300K.bfilt.narrowPeak.gz,narrow ON2_Treg,ON,2,ON2_Treg_S10_L001_R1_001.trim.merged.bam,ON2_Treg_S10_L001_R1_001.trim.merged.nodup.no_chrM_MT.tn5.pval0.01.300K.bfilt.narrowPeak.gz,narrow 91F_Treg,OFF,1,91F_Treg_S28_L002_R1_001.trim.merged.bam,91F_Treg_S28_L002_R1_001.trim.merged.nodup.no_chrM_MT.tn5.pval0.01.300K.bfilt.narrowPeak.gz,narrow 96F_Treg,OFF,2,96F_Treg_S29_L002_R1_001.trim.merged.bam,96F_Treg_S29_L002_R1_001.trim.merged.nodup.no_chrM_MT.tn5.pval0.01.300K.bfilt.narrowPeak.gz,narrow

Here is the code for DiffBind Analysis

library(DiffBind)

treg=dba(sampleSheet="PS_Treg_atacseq-1.csv")
treg=dba.count(treg,bUseSummarizeOverlaps=TRUE)
treg=dba.contrast(treg,categories=DBA_FACTOR,minMembers=2)
treg=dba.analyze(treg,method=DBA_ALL_METHODS)
treg.DB=dba.report(treg)
saveRDS(treg.DB,"treg.DB.rds")

I get the following error when I run the code above:

Error: Error processing one or more read files. Check warnings().
In addition: Warning messages:
1: In mclapply(arglist, fn, ..., mc.preschedule = TRUE, mc.allow.recursive = TRUE) :
  all scheduled cores encountered errors in user code
2:   subsetting operation 
3:   subsetting operation 
4:   subsetting operation 
5:   subsetting operation 
Execution halted
 [1] DiffBind_3.0.13             SummarizedExperiment_1.20.0
 [3] Biobase_2.50.0              MatrixGenerics_1.2.1       
 [5] matrixStats_0.58.0          GenomicRanges_1.42.0       
 [7] GenomeInfoDb_1.26.2         IRanges_2.24.1             
 [9] S4Vectors_0.28.1            BiocGenerics_0.36.0        

loaded via a namespace (and not attached):
  [1] amap_0.8-18              colorspace_2.0-0         rjson_0.2.20            
  [4] hwriter_1.3.2            ellipsis_0.3.1           XVector_0.30.0          
  [7] rstudioapi_0.13          ggrepel_0.8.2            bit64_4.0.5             
 [10] mvtnorm_1.1-1            apeglm_1.12.0            AnnotationDbi_1.52.0    
 [13] splines_4.0.0            jsonlite_1.7.2           Rsamtools_2.6.0         
 [16] annotate_1.68.0          ashr_2.2-47              GO.db_3.11.4            
 [19] dbplyr_2.1.0             png_0.1-7                GreyListChIP_1.22.0     
 [22] pheatmap_1.0.12          graph_1.68.0             compiler_4.0.0          
 [25] httr_1.4.2               GOstats_2.56.0           backports_1.2.1         
 [28] assertthat_0.2.1         Matrix_1.2-18            limma_3.46.0            
 [31] prettyunits_1.1.1        tools_4.0.0              coda_0.19-4             
 [34] gtable_0.3.0             glue_1.4.2               GenomeInfoDbData_1.2.4  
 [37] Category_2.56.0          systemPipeR_1.24.3       dplyr_1.0.4             
 [40] rsvg_2.1                 batchtools_0.9.15        rappdirs_0.3.3          
 [43] V8_3.2.0                 ShortRead_1.48.0         Rcpp_1.0.6              
 [46] bbmle_1.0.23.1           vctrs_0.3.6              Biostrings_2.58.0       
 [49] rtracklayer_1.50.0       stringr_1.4.0            irlba_2.3.3             
 [52] lifecycle_1.0.0          gtools_3.8.2             XML_3.99-0.5            
 [55] edgeR_3.30.3             MASS_7.3-51.6            zlibbioc_1.36.0         
 [58] scales_1.1.1             BSgenome_1.58.0          VariantAnnotation_1.34.0
 [61] hms_1.0.0                RBGL_1.66.0              RColorBrewer_1.1-2      
 [64] yaml_2.2.1               curl_4.3                 memoise_1.1.0           
 [67] ggplot2_3.3.3            emdbook_1.3.12           bdsmatrix_1.3-4         
 [70] biomaRt_2.44.1           SQUAREM_2021.1           latticeExtra_0.6-29     
 [73] stringi_1.5.3            RSQLite_2.2.0            genefilter_1.72.1       
 [76] checkmate_2.0.0          GenomicFeatures_1.42.1   caTools_1.18.1          
 [79] BiocParallel_1.24.1      DOT_0.1                  truncnorm_1.0-8         
 [82] rlang_0.4.10             pkgconfig_2.0.3          bitops_1.0-6            
 [85] invgamma_1.1             lattice_0.20-41          purrr_0.3.4             
 [88] GenomicAlignments_1.24.0 bit_4.0.4                tidyselect_1.1.0        
 [91] GSEABase_1.52.1          AnnotationForge_1.32.0   plyr_1.8.6              
 [94] magrittr_2.0.1           R6_2.5.0                 gplots_3.1.1            
 [97] generics_0.1.0           base64url_1.4            DelayedArray_0.16.1     
[100] DBI_1.1.1                pillar_1.4.6             withr_2.4.1             
[103] mixsqp_0.3-43            survival_3.2-7           RCurl_1.98-1.2          
[106] tibble_3.0.3             crayon_1.4.1             KernSmooth_2.23-17      
[109] BiocFileCache_1.14.0     jpeg_0.1-8.1             progress_1.2.2          
[112] locfit_1.5-9.4           grid_4.0.0               data.table_1.14.0       
[115] blob_1.2.1               Rgraphviz_2.34.0         digest_0.6.27           
[118] xtable_1.8-4             numDeriv_2016.8-1.1      brew_1.0-6              
[121] openssl_1.4.3            munsell_0.5.0            askpass_1.1

I also used bParallel=FALSE in count and received the following error

Computing summits...
Sample: 11_12_Treg/align/rep1/11_Treg_S7_L001_R1_001.trim.merged.nodup.no_chrM_MT.bam125 
Sample: 11_12_Treg/align/rep2/12_Treg_S8_L001_R1_001.trim.merged.nodup.no_chrM_MT.bam125 
Sample: 51F_ON2_Treg/align/rep1/51F_Treg_S9_L001_R1_001.trim.merged.nodup.no_chrM_MT.bam125 
Sample: 51F_ON2_Treg/align/rep2/ON2_Treg_S10_L001_R1_001.trim.merged.nodup.no_chrM_MT.bam125 
Sample: 91F_96F_Treg/align/rep1/91F_Treg_S28_L002_R1_001.trim.merged.nodup.no_chrM_MT.bam125 
Sample: 91F_96F_Treg/align/rep2/96F_Treg_S29_L002_R1_001.trim.merged.nodup.no_chrM_MT.bam125 
Re-centering peaks...
Reads will be counted as Paired-end.
Sample: 11_12_Treg/align/rep1/11_Treg_S7_L001_R1_001.trim.merged.nodup.no_chrM_MT.bam125 
Error in validObject(x) : invalid class “GAlignments” object: 
    'mcols(x)' is not parallel to 'x'
Calls: dba.count ... tryCatch -> tryCatchList -> tryCatchOne -> <Anonymous>
In addition: Warning message:
In .make_GAlignmentPairs_from_GAlignments(gal, strandMode = strandMode,  :
    34113122 alignments with ambiguous pairing were dumped.
    Use 'getDumpedAlignments()' to retrieve them from the dump environment.
Execution halted

Could you please help me understand the cause of this error? I did not get any errors while aligning data.

ATACSeq DiffBind • 292 views
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Entering edit mode

The DiffBind maintainer will probably answer. In the meantime what is the output of warnings(), and at which step of the listed ones does it crash?

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crashes at the count step.

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Entering edit mode

I re-ran the analysis with just the WT and ON samples and I get similar kind of errors:

Error: Error processing one or more read files. Check warnings(). In addition: Warning messages: 1: In mclapply(arglist, fn, ..., mc.preschedule = TRUE, mc.allow.recursive = TRUE) : all scheduled cores encountered errors in user code 2: 'mcols(x)' is not parallel to 'x' 3: 'mcols(x)' is not parallel to 'x' 4: 'mcols(x)' is not parallel to 'x' 5: 'mcols(x)' is not parallel to 'x' Execution halted

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Entering edit mode
Rory Stark ★ 4.1k
@rory-stark-5741
Last seen 1 day ago
CRUK, Cambridge, UK

There error is coming out of the GenomicAlignments library -- there are ambiguities with the paired-end alignments in the bam files. Have a a look at the help page:

library(GenomicAlignments)
?findMateAlignment

You can also set bUseSummarizeOverlaps=FALSE in the call to dba.count() to see if that works (it will count each read separately).

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