Hi,
I have a question about whether I should set the sizeFactors to 1 prior to using DESeq2 on the counts obtained from derfinder. I have normalized bigwig files ( using deepTools ), which I use as input to derfinder. Following the instructions in the users guide : lcolladotor.github.io/derfinder/articles/derfinder-users-guide.html#expressed-regions-level-1
My doubt is that if the counts obtained by derfinder are already from normalized bigwig files, whether I should set the sizeFactors to 1 before running the DESeq function.
Thank you,
Debayan
Code should be placed in three backticks as shown below
# include your problematic code here with any corresponding output
# please also include the results of running the following in an R session
## Determine the files to use and fix the names
files_RZ <- list.files(path="C:/Users/rnaseq_RZ/", pattern=".*bw$",full.names=T)
names(files_RZ) <- gsub(".deepTools.bw", "", basename(files_RZ))
## Load the data from disk -- On Windows you have to load data from Bam files
chrs=paste("chr",c(1:22,"X","Y","M"),sep="")
fullCov_RZ <- fullCoverage(files = files_RZ, chrs=chrs, verbose = FALSE)
## Get the region matrix of Expressed Regions (ERs)
regionMat_RZ <- regionMatrix(fullCov_RZ, cutoff = 30, L = 100, verbose = FALSE)
# Combine into a single one
## First extract the data
regs <- unlist(GRangesList(lapply(regionMat_RZ, '[[', 'regions')))
covMat <- do.call(rbind, lapply(regionMat_RZ, '[[', 'coverageMatrix'))
## Force the names to match
names(regs) <- rownames(covMat) <- seq_len(length(regs))
## Combine into a list (not really needed)
mergedRegionMat_RZ <- list('regions' = regs, 'coverageMatrix' = covMat)
## Round matrix
counts_RZ <- round(mergedRegionMat_RZ$coverageMatrix)
##Design
coldata=data.frame(factor(c("1","2","2","2","1","1","2","1","3","3","3","3")))
colnames(coldata)="condition"
rownames(coldata)=colnames(counts_RZ)
dds_RZ <- DESeqDataSetFromMatrix(countData = counts_RZ,
colData = coldata,
design = ~ condition)
dds2_RZ <- DESeq(dds_RZ)
sessionInfo( )
R version 4.0.3 (2020-10-10)
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
[3] 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] reshape2_1.4.4 forcats_0.5.0 purrr_0.3.4
[4] readr_1.4.0 tidyr_1.1.2 tidyverse_1.3.0
[7] derfinder_1.24.2 ggrepel_0.8.2 DESeq2_1.30.0
[10] SummarizedExperiment_1.20.0 Biobase_2.50.0 MatrixGenerics_1.2.0
[13] matrixStats_0.57.0 GenomicRanges_1.42.0 GenomeInfoDb_1.26.2
[16] IRanges_2.24.0 S4Vectors_0.28.1 BiocGenerics_0.36.0
[19] leaflet_2.0.4.1 stringr_1.4.0 dplyr_1.0.2
[22] ggplot2_3.3.3 tibble_3.0.4 RColorBrewer_1.1-2
[25] shiny_1.6.0
loaded via a namespace (and not attached):
[1] readxl_1.3.1 backports_1.2.0 Hmisc_4.5-0 BiocFileCache_1.14.0
[5] plyr_1.8.6 lazyeval_0.2.2 sp_1.4-5 splines_4.0.3
[9] BiocParallel_1.24.1 crosstalk_1.1.1 digest_0.6.27 foreach_1.5.1
[13] ensembldb_2.14.1 htmltools_0.5.1.1 viridis_0.5.1 leaflet.providers_1.9.0
[17] magrittr_2.0.1 checkmate_2.0.0 memoise_2.0.0 BSgenome_1.58.0
[21] cluster_2.1.0 Biostrings_2.58.0 annotate_1.68.0 modelr_0.1.8
[25] askpass_1.1 prettyunits_1.1.1 jpeg_0.1-8.1 colorspace_2.0-0
[29] rvest_0.3.6 blob_1.2.1 rappdirs_0.3.1 haven_2.3.1
[33] xfun_0.25 crayon_1.3.4 RCurl_1.98-1.2 jsonlite_1.7.1
[37] genefilter_1.72.0 survival_3.2-7 VariantAnnotation_1.36.0 iterators_1.0.13
[41] glue_1.4.2 gtable_0.3.0 zlibbioc_1.36.0 XVector_0.30.0
[45] DelayedArray_0.16.0 scales_1.1.1 DBI_1.1.0 rngtools_1.5
[49] derfinderHelper_1.24.1 Rcpp_1.0.5 viridisLite_0.3.0 xtable_1.8-4
[53] progress_1.2.2 htmlTable_2.2.1 bumphunter_1.32.0 foreign_0.8-80
[57] bit_4.0.4 Formula_1.2-4 htmlwidgets_1.5.3 httr_1.4.2
[61] ellipsis_0.3.1 pkgconfig_2.0.3 XML_3.99-0.5 farver_2.0.3
[65] nnet_7.3-14 sass_0.4.0 dbplyr_2.0.0 locfit_1.5-9.4
[69] tidyselect_1.1.0 labeling_0.4.2 rlang_0.4.11 later_1.2.0
[73] AnnotationDbi_1.52.0 cellranger_1.1.0 munsell_0.5.0 tools_4.0.3
[77] cachem_1.0.5 cli_3.0.1 generics_0.1.0 RSQLite_2.2.1
[81] broom_0.7.6 fastmap_1.1.0 yaml_2.2.1 fs_1.5.0
[85] knitr_1.33 bit64_4.0.5 AnnotationFilter_1.14.0 doRNG_1.8.2
[89] mime_0.9 xml2_1.3.2 biomaRt_2.46.0 compiler_4.0.3
[93] rstudioapi_0.13 curl_4.3 png_0.1-7 reprex_0.3.0
[97] geneplotter_1.68.0 bslib_0.2.5.1 stringi_1.5.3 GenomicFeatures_1.42.1
[101] GenomicFiles_1.26.0 lattice_0.20-41 ProtGenerics_1.22.0 Matrix_1.2-18
[105] vctrs_0.3.5 pillar_1.4.7 lifecycle_1.0.0 jquerylib_0.1.4
[109] data.table_1.13.4 bitops_1.0-6 raster_3.4-13 qvalue_2.22.0
[113] httpuv_1.6.1 rtracklayer_1.49.5 R6_2.5.0 latticeExtra_0.6-29
[117] promises_1.2.0.1 gridExtra_2.3 codetools_0.2-16 assertthat_0.2.1
[121] openssl_1.4.3 withr_2.4.2 GenomicAlignments_1.26.0 Rsamtools_2.6.0
[125] GenomeInfoDbData_1.2.4 hms_0.5.3 grid_4.0.3 rpart_4.1-15
[129] lubridate_1.7.9.2 base64enc_0.1-3 tinytex_0.33
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