Hi, I did an analysis using DiffBind in June 2022 with no errors. I restarted the project last week after updating R and DiffBind and now the normalization process does not work when the blacklist argument = TRUE.
1) My command with an error message is:
peaks.400 <- dba.analyze(peaks.400, method=DBA_ALL_METHODS_BLOCK)
2) Commands performed just before:
peaks <- dba(sampleSheet = sample.table, minOverlap=1, config=data.frame(RunParallel=FALSE, reportInit="DBA", DataType=DBA_DATA_GRANGES, AnalysisMethod=DBA_DESEQ2,fragmentSize=160, bCorPlot=FALSE, th=0.1, bUsePval=FALSE))
peaks.400 <- dba.count(peaks, summits = 200)
peaks.400 <- dba.normalize(peaks.400, offsets = TRUE)
peaks.400 <- dba.contrast(peaks.400, design = FALSE, categories = DBA_TREATMENT, block = DBA_REPLICATE, minMembers = 2)
`
The error returned was:
Applying Blacklist/Greylists...
Genome detected: Mmusculus.UCSC.mm10
Applying blacklist...
Removed: 944 of 38060 intervals.
Re-normalizing...
Blacklist error: Error: Invalid normalization
Unable to apply Blacklist/Greylist.
3) I tried to remove the blacklist:
peaks.400B <- dba.analyze(peaks.400, method=DBA_ALL_METHODS_BLOCK, bBlacklist = FALSE)
And then the problem disappeared:
Normalize edgeR with defaults...
Analyzing...
edgeR multi-factor analysis.
edgeR multi-factor analysis.
edgeR multi-factor analysis.
converting counts to integer mode
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
converting counts to integer mode
DESeq2 multi-factor analysis
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
converting counts to integer mode
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
converting counts to integer mode
DESeq2 multi-factor analysis
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
converting counts to integer mode
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
converting counts to integer mode
DESeq2 multi-factor analysis
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
4) Finally, I tried:
peaks.400 <- dba.blacklist(peaks.400, blacklist=DBA_BLACKLIST_MM10, greylist= FALSE)
But got the following error message:
Genome detected: Mmusculus.UCSC.mm10
Applying blacklist...
Removed: 944 of 38060 intervals.
Re-normalizing...
Error: Invalid normalization
5) Like I mentioned above, with an earlier R version (R version 4.1.2 (2021-11-01) -- "Bird Hippie") and DiffBind version (not sure which one) on exactly the same BAM files with the same code I did not have this problem.
However, I would like to compare with and without blacklist applied to see the validity of my results. Any idea what the problem could be?
Thanks a lot for your help.
sessionInfo( )
R version 4.2.2 (2022-10-31)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Monterey 12.3.1
Matrix products: default
LAPACK: /Library/Frameworks/R.framework/Versions/4.2/Resources/lib/libRlapack.dylib
locale:
[1] en_CA.UTF-8/en_CA.UTF-8/en_CA.UTF-8/C/en_CA.UTF-8/en_CA.UTF-8
attached base packages:
[1] parallel stats4 stats graphics grDevices utils datasets methods base
other attached packages:
[1] GreyListChIP_1.30.0 BSgenome.Mmusculus.UCSC.mm10_1.4.3 BSgenome_1.66.1
[4] BiocManager_1.30.19 csaw_1.32.0 rtracklayer_1.58.0
[7] DiffBind_3.8.0 minfi_1.44.0 bumphunter_1.40.0
[10] locfit_1.5-9.6 iterators_1.0.14 foreach_1.5.2
[13] Biostrings_2.66.0 XVector_0.38.0 SummarizedExperiment_1.28.0
[16] Biobase_2.58.0 MatrixGenerics_1.10.0 matrixStats_0.62.0
[19] GenomicRanges_1.50.1 GenomeInfoDb_1.34.3 IRanges_2.32.0
[22] S4Vectors_0.36.0 BiocGenerics_0.44.0
loaded via a namespace (and not attached):
[1] BiocFileCache_2.6.0 plyr_1.8.7 splines_4.2.2 BiocParallel_1.32.1
[5] ggplot2_3.4.0 amap_0.8-19 digest_0.6.30 invgamma_1.1
[9] htmltools_0.5.3 SQUAREM_2021.1 fansi_1.0.3 magrittr_2.0.3
[13] memoise_2.0.1 tzdb_0.3.0 limma_3.54.0 readr_2.1.3
[17] annotate_1.76.0 systemPipeR_2.4.0 bdsmatrix_1.3-6 askpass_1.1
[21] siggenes_1.72.0 prettyunits_1.1.1 jpeg_0.1-9 colorspace_2.0-3
[25] apeglm_1.20.0 ggrepel_0.9.2 blob_1.2.3 rappdirs_0.3.3
[29] dplyr_1.0.10 crayon_1.5.2 RCurl_1.98-1.9 genefilter_1.80.0
[33] GEOquery_2.66.0 survival_3.4-0 glue_1.6.2 gtable_0.3.1
[37] zlibbioc_1.44.0 DelayedArray_0.24.0 Rhdf5lib_1.20.0 HDF5Array_1.26.0
[41] scales_1.2.1 mvtnorm_1.1-3 edgeR_3.40.0 DBI_1.1.3
[45] rngtools_1.5.2 Rcpp_1.0.9 emdbook_1.3.12 xtable_1.8-4
[49] progress_1.2.2 bit_4.0.4 mclust_6.0.0 preprocessCore_1.60.0
[53] truncnorm_1.0-8 metapod_1.6.0 htmlwidgets_1.5.4 httr_1.4.4
[57] gplots_3.1.3 RColorBrewer_1.1-3 ellipsis_0.3.2 pkgconfig_2.0.3
[61] reshape_0.8.9 XML_3.99-0.12 deldir_1.0-6 dbplyr_2.2.1
[65] utf8_1.2.2 tidyselect_1.2.0 rlang_1.0.6 AnnotationDbi_1.60.0
[69] munsell_0.5.0 tools_4.2.2 cachem_1.0.6 cli_3.4.1
[73] generics_0.1.3 RSQLite_2.2.18 stringr_1.4.1 fastmap_1.1.0
[77] yaml_2.3.6 bit64_4.0.5 beanplot_1.3.1 caTools_1.18.2
[81] scrime_1.3.5 purrr_0.3.5 KEGGREST_1.38.0 nlme_3.1-160
[85] doRNG_1.8.2 sparseMatrixStats_1.10.0 nor1mix_1.3-0 xml2_1.3.3
[89] biomaRt_2.54.0 compiler_4.2.2 rstudioapi_0.14 filelock_1.0.2
[93] curl_4.3.3 png_0.1-7 geneplotter_1.76.0 tibble_3.1.8
[97] stringi_1.7.8 GenomicFeatures_1.50.2 lattice_0.20-45 Matrix_1.5-1
[101] multtest_2.54.0 vctrs_0.5.0 pillar_1.8.1 lifecycle_1.0.3
[105] rhdf5filters_1.10.0 irlba_2.3.5.1 data.table_1.14.4 bitops_1.0-7
[109] R6_2.5.1 BiocIO_1.8.0 latticeExtra_0.6-30 hwriter_1.3.2.1
[113] ShortRead_1.56.0 KernSmooth_2.23-20 codetools_0.2-18 MASS_7.3-58.1
[117] gtools_3.9.3 assertthat_0.2.1 rhdf5_2.42.0 DESeq2_1.38.0
[121] openssl_2.0.4 rjson_0.2.21 GenomicAlignments_1.34.0 Rsamtools_2.14.0
[125] GenomeInfoDbData_1.2.9 hms_1.1.2 quadprog_1.5-8 grid_4.2.2
[129] coda_0.19-4 tidyr_1.2.1 base64_2.0.1 DelayedMatrixStats_1.20.0
[133] ashr_2.2-54 illuminaio_0.40.0 mixsqp_0.3-43 bbmle_1.0.25
[137] numDeriv_2016.8-1.1 interp_1.1-3 restfulr_0.0.15
Thanks a lot!
Hi, i have the same problem in DiffBind 3.10.1, how should i fix it? My command with an error message is
dbObj <- dba.analyze(dbObj)
, the error retrurns below at the step of re-normalizingBlacklist error: Error: Invalid normalization
My R version is 4.3.1, Thanks a lot for help