How to set $control.subtract to FALSE in DiffBind?
Entering edit mode
ksong ▴ 10
Last seen 9 weeks ago
United States

Dear friends,
I have been using diffbind to get RLE/TMM normalized counts. I have noticed that the RLE/TMM normalization is performed based on reads subtracted by control reads. How to turn off the subtraction? I have tried following by setting bSubControl = F, but it does not work.


h3k27ac_samples <- read.csv("h3k27ac.csv") #use sorted bam
h3k27ac <- dba(sampleSheet=h3k27ac_samples)
h3k27ac <- dba.blacklist(h3k27ac, blacklist =T , greylist = F)
h3k27ac <- dba.count(h3k27ac, bSubControl = F) #here the default score is library size norm

#get raw counts
h3k27ac <- dba.count(h3k27ac, peaks=NULL, score=DBA_SCORE_READS, bSubControl = F)
h3k27ac_count_raw <- dba.peakset(h3k27ac, bRetrieve=TRUE)

#get library size normalized reads
h3k27ac <- dba.normalize(h3k27ac) #default normalize = DBA_NORM_DEFAULT meaning normalize=DBA_NORM_LIB library=DBA_LIBSIZE_FULL background=FALSE
h3k27ac <- dba.count(h3k27ac, peaks = NULL, score = DBA_SCORE_NORMALIZED, bSubControl = F) 
h3k27ac_count_lib_norm = dba.peakset(h3k27ac, bRetrieve=TRUE) 
h3k27ac_lib_norm_info = dba.normalize(h3k27ac, bRetrieve = T)

# get RLE normalized counts
h3k27ac <- dba.normalize(h3k27ac, normalize = DBA_NORM_RLE) # normalize = DBA_NORM_RLE
h3k27ac <- dba.count(h3k27ac, peaks = NULL, score = DBA_SCORE_NORMALIZED, bSubControl = F) 
h3k27ac_count_rle_norm = dba.peakset(h3k27ac, bRetrieve =TRUE) 
h3k27ac_rle_norm_info <- dba.normalize(h3k27ac, normalize = DBA_NORM_RLE, bRetrieve = T)

# get TMM normalized counts
h3k27ac <- dba.normalize(h3k27ac, normalize = DBA_NORM_TMM) # normalize = DBA_NORM_TMM
h3k27ac <- dba.count(h3k27ac, peaks = NULL, score = DBA_SCORE_NORMALIZED, bSubControl = F) 
h3k27ac_count_tmm_norm = dba.peakset(h3k27ac, bRetrieve=TRUE) 
h3k27ac_tmm_norm_info <- dba.normalize(h3k27ac, normalize = DBA_NORM_TMM, bRetrieve = T)

None of h3k27ac_lib_norm_info, h3k27ac_rle_norm_info and h3k27ac_tmm_norm_info shows $control.subtract = F for example

[1] TRUE

Here is my sessionInfo

> sessionInfo()
R version 4.2.0 (2022-04-22)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.3 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/blas/
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/

 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C               LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8     LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                  LC_ADDRESS=C               LC_TELEPHONE=C             LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

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

other attached packages:
 [1] forcats_0.5.1               stringr_1.4.0               dplyr_1.0.9                 purrr_0.3.4                 readr_2.1.2                 tidyr_1.2.0                
 [7] tibble_3.1.7                ggplot2_3.3.6               tidyverse_1.3.1             DiffBind_3.6.1              SummarizedExperiment_1.26.1 Biobase_2.56.0             
[13] MatrixGenerics_1.8.0        matrixStats_0.62.0          GenomicRanges_1.48.0        GenomeInfoDb_1.32.2         IRanges_2.30.0              S4Vectors_0.34.0           
[19] BiocGenerics_0.42.0        

loaded via a namespace (and not attached):
  [1] amap_0.8-18              colorspace_2.0-3         rjson_0.2.21             hwriter_1.3.2.1          ellipsis_0.3.2           XVector_0.36.0          
  [7] fs_1.5.2                 rstudioapi_0.13          bit64_4.0.5              ggrepel_0.9.1            AnnotationDbi_1.58.0     fansi_1.0.3             
 [13] mvtnorm_1.1-3            apeglm_1.18.0            lubridate_1.8.0          xml2_1.3.3               splines_4.2.0            cachem_1.0.6            
 [19] geneplotter_1.74.0       jsonlite_1.8.0           Rsamtools_2.12.0         annotate_1.74.0          broom_0.8.0              ashr_2.2-54             
 [25] dbplyr_2.1.1             png_0.1-7                GreyListChIP_1.28.1      compiler_4.2.0           httr_1.4.3               backports_1.4.1         
 [31] assertthat_0.2.1         Matrix_1.4-1             fastmap_1.1.0            limma_3.52.1             cli_3.3.0                htmltools_0.5.2         
 [37] tools_4.2.0              coda_0.19-4              gtable_0.3.0             glue_1.6.2               GenomeInfoDbData_1.2.8   systemPipeR_2.2.2       
 [43] ShortRead_1.54.0         Rcpp_1.0.8.3             bbmle_1.0.25             cellranger_1.1.0         vctrs_0.4.1              Biostrings_2.64.0       
 [49] rtracklayer_1.56.0       rvest_1.0.2              lifecycle_1.0.1          irlba_2.3.5              restfulr_0.0.13          gtools_3.9.2.1          
 [55] XML_3.99-0.9             edgeR_3.38.1             zlibbioc_1.42.0          MASS_7.3-57              scales_1.2.0             BSgenome_1.64.0         
 [61] hms_1.1.1                parallel_4.2.0           RColorBrewer_1.1-3       yaml_2.3.5               memoise_2.0.1            emdbook_1.3.12          
 [67] bdsmatrix_1.3-4          RSQLite_2.2.14           latticeExtra_0.6-29      stringi_1.7.6            SQUAREM_2021.1           genefilter_1.78.0       
 [73] BiocIO_1.6.0             caTools_1.18.2           BiocParallel_1.30.2      truncnorm_1.0-8          rlang_1.0.2              pkgconfig_2.0.3         
 [79] bitops_1.0-7             lattice_0.20-45          invgamma_1.1             GenomicAlignments_1.32.0 htmlwidgets_1.5.4        bit_4.0.4               
 [85] tidyselect_1.1.2         DESeq2_1.36.0            plyr_1.8.7               magrittr_2.0.3           R6_2.5.1                 gplots_3.1.3            
 [91] generics_0.1.2           DelayedArray_0.22.0      DBI_1.1.2                pillar_1.7.0             haven_2.5.0              withr_2.5.0             
 [97] survival_3.3-1           KEGGREST_1.36.0          RCurl_1.98-1.6           mixsqp_0.3-43            modelr_0.1.8             crayon_1.5.1            
[103] KernSmooth_2.23-20       utf8_1.2.2               tzdb_0.3.0               jpeg_0.1-9               locfit_1.5-9.5           grid_4.2.0              
[109] readxl_1.4.0             blob_1.2.3               reprex_2.0.1             digest_0.6.29            xtable_1.8-4             numDeriv_2016.8-1.1     
[115] munsell_0.5.0
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