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.
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?crashes at the count step.
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